Methods for enhancing nucleic acid hybridization

ABSTRACT

A composition comprising an oligonucleotide having the structure 5′-Y 1 -L 1 -X-L 2 -Y 2 -3′. Y 1  comprises a sequence of one or more DNA or RNA nucleotides, including a first nucleotide N 1  having a 3′ phosphate covalently linked to L 1 . Y 2  comprises a sequence of one or more DNA or RNA nucleotides, including a second nucleotide N 2  having a 5′ phosphate covalently linked to L 2 . L 1  and L 2  each independently are a direct bond or a C 1 -C 7  alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl group. X is 
                         
R 1  is a hydrogen or a C 1 -C 8  alkyl. M is a label or ligand comprising a fused polycyclic aromatic moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 61/318,043 filed Mar. 26, 2010, the content of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The sequence listing is filed with the application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “ASFILED_Sequence_US00” was created on Mar. 25, 2011, and is 49,585bytes in size.

FIELD OF THE DISCLOSURE

This disclosure pertains to novel oligonucleotide compounds withimproved hybridization properties. Methods and reagents are providedwhich allow internal labeling of oligonucleotides by insertion of labelsbetween adjacent residues without destabilizing the duplex. Because thenucleotide bases are not modified, such labeling groups can beintroduced into any sequence. In some embodiments, such modificationsincrease duplex stability. In some embodiments, the labeling group is afluorescence quencher. The disclosure further relates to the design offluorescently labeled oligonucleotide probes with multiple quenchingdyes capable of very efficient fluorescence quenching over a broadspectral range.

BACKGROUND

Fluorescent energy transfer probes are an important tool in geneticanalysis. These probes, also known as dual-labeled probes (DLPs) orself-quenching probes, are generally comprised of a fluorescent donor (afluorophore) and a quencher linked to an oligonucleotide. This basicdesign, wherein a signal change is detected once the probe hybridizes toits intended target, is used in a variety of biological applications.

One method for detecting hybridization using fluorophores and quenchersis to link fluorescent donors and quenchers to a single oligonucleotidesuch that there is a detectable difference in fluorescence when theoligonucleotide is unhybridized as compared to when it is hybridized toits complementary sequence. In so-called molecular beacons, a partiallyself-complementary oligonucleotide is designed to form a hairpin and islabeled with a fluorescent donor at one end of the molecule and aquencher at the other end (U.S. Pat. No. 5,925,517). Intramolecularannealing to form the hairpin brings the donor and quencher into closeproximity for fluorescent quenching to occur. Intermolecular annealingof such an oligonucleotide to a target sequence disrupts the hairpin,which increases the distance between the donor and quencher and resultsin a detectable increase in the fluorescent signal of the donor.

Oligonucleotides are not required to form a hairpin structure for thismethod to work efficiently. The fluorophore and quencher can be placedon an oligonucleotide such that when it is unhybridized and in a randomcoil conformation, the quencher is able to quench fluorescence from thefluorophore (U.S. Pat. No. 5,538,848). Once the oligonucleotidehybridizes to a complementary nucleotide sequence it becomes moreextended and the distance between the fluorophore and quencher isincreased, resulting in reduced quenching and increased fluorescence.

Oligonucleotides labeled in a similar manner can also be used to monitorthe kinetics of PCR amplification. In one version of this method,commonly known as a 5′-nuclease cleavage or hydrolysis assay, anoligonucleotide probe is designed to hybridize to the target sequence onthe 3′ side (“downstream”) of one of the amplification primers. DuringPCR, the 5′-3′ exonuclease activity of the DNA polymerase digests the 5′end of the probe thereby separating the fluorophore from the quencher.The fluorescence intensity of the sample increases as an increasingnumber of probe molecules are digested during the course ofamplification (U.S. Pat. No. 5,210,015).

DLPs find use in other molecular/cellular biology and diagnostic assays,such as in end-point PCR, in situ hybridizations, in vivo DNA and RNAspecies detection, single nucleotide polymorphism (SNPs) analysis,enzyme assays, and in vivo and in vitro whole cell assays (see Dirks andTanke, Biotechniques 2006, 40:489-486; Bustin, Journal of MolecularEndocrinology 2002, 29:23-39; Mackay, Clin Microbiol Infect, 2004,10:190-212).

In one mechanism of fluorescence quenching termed ground statequenching, the fluorophore and the quencher associate to form a groundstate complex which is not fluorescent. For ground state quenching tooccur there need not be spectral overlap between the fluorophore and thequencher.

The most common mechanism of fluorescent quenching is fluorescenceresonance energy transfer (FRET). In FRET, energy transfer occursthrough space by dipolar coupling between the fluorophore and quencherand requires that there be overlap between the emission spectrum of thefluorescent donor and the absorbance spectrum of the quencher. Thisrequirement complicates the design of probes that utilize FRET becausequenchers are limited in their effective wavelength range. For example,the quencher known as BHQ-1, which absorbs light in the wavelength rangeof about 500-550 nm, quenches fluorescent light emitted by fluorescein,which fluoresces maximally at about 520 nm, but is of limited utilityfor Texas Red (emission maximum=615) or Cy5 (emission maximum=670). Incontrast, the quencher BHQ-3, which absorbs light in the wavelengthrange of about 650-700 nm is almost completely ineffective at quenchingfluorescein but is effective at quenching Cy5. In general, the number ofquenchers that are known to be capable of quenching the fluorescence ofany given fluorophore is limited.

Although fluorescent dyes themselves can be employed to quenchfluorescence from other dyes, preferred quenchers will not fluoresce (orminimally fluoresce) so that background fluorescence is minimized. Thesequenchers are commonly referred to as dark quenchers. Dark quenchersallow for an increased signal to noise ratio in assays that employ DLPs,resulting in increased sensitivity. In addition, the lack of secondaryfluorescence facilitates the use of additional fluorophores inmultiplexed assay formats which utilize multiple distinct probes eachcontaining a different fluorophore. If a quencher emitted light in acertain region, then additional probes could not bear fluorophores thatemit light in that same region.

A number of factors are considered in designing a self-quenching probe.These include the ease of synthesis, the compatibility of thefluorophore and quencher, duplex stability, and the specificity of theprobe in hybridizing to the intended target.

Duplex stability between complementary nucleic acid molecules isfrequently expressed as the “melting temperature”, T_(m), of the duplex.Roughly speaking, the T_(m) indicates the temperature at which a duplexnucleic acid dissociates into two single strands. Nucleic acidhybridization is generally performed at a temperature slightly below theT_(m), so that hybridization between a probe or primer and its targetnucleic acid is optimized, while minimizing non-specific hybridizationof the probe or primer to other, non-target nucleic acids. Duplexstability and T_(m) are also important in applications, such as PCR,where thermocycling may be involved. During such thermocycling meltingsteps, it is important that the sample temperature be raisedsufficiently above the T_(m) so that duplexes of the target nucleic acidand its complement are dissociated. In subsequent steps of reannealing,however, the temperature must be brought sufficiently below the T_(m)that duplexes of the target nucleic acid and primer are able to form,while still remaining high enough to avoid non-specific hybridizationevents. For a general discussion, see Rychlik et al., Nucleic AcidsResearch 1990, 18:6409-6412.

Shorter oligonucleotides can help increase the specificity of a primeror probe, allowing for the discrimination of even a single mismatchbetween the probe and a potential complementary target. The shorter theoligonucleotide, the greater the effect of a single-base mismatch onduplex stability. However, the disadvantage of using such shortoligonucleotides is that they hybridize weakly, even to a perfectlycomplementary sequence, and thus must be used at lower temperatures,which are unfavorable for reactions that use thermal stable enzymes,such as PCR. Certain modified nucleosides such as locked nucleic acids(LNAs) (U.S. Pat. No. 7,060,809) and C5-propynyl pyrimidines (U.S. Pat.No. 5,484,908) can be incorporated into oligonucleotides to increaseduplex stability. Many nucleoside analogs, however, especially thosehaving bulkier substituents attached to the base, are destabilizing. Forexample, fluorescein-dT can destabilize a duplex by up to 4° C.(Bioorganic & Medicinal Chemistry Letters, 13:2785-2788 2003).

Modified nucleosides employed to increase Tm are typically placedinternally within an oligonucleotide sequence replacing a natural base.In contrast, non-nucleoside substituents when introduced internallywithin an oligonucleotide, either as a replacement for a base or as aninsertion between bases, generally interfere with hybridization. Forexample, insertion of an abasic fluorescein group into anoligonucleotide has been observed to destabilize a duplex by 2-4° C.(DNA Seq. 4:135-141, 1993).

There are several classes of compounds that are known to increasebinding affinity between complementary nucleic acid strands. One classis major groove binders, which includes proteins or ligands that bind tothe major groove (the wider groove around a DNA helix). A second class,minor groove binders (MGBs), include non-covalently bound and covalentlybound compounds. Because the minor groove of a helix is narrower in A-Trich regions, some noncovalently bound MBGs recognize the shape of thehelix and preferably bind to specific sequence regions. For example,netropsin and distamycin preferably bind to A-T regions (see Bailly andHenichart, ACS, vol. 2, 379-393 (1991). Covalently bound MGBs (U.S. Pat.No. 6,084,102) are typically linked to the 5′ or 3′ end ofoligonucleotides (U.S. Pat. App. 2009/0259030) and are known to increasebinding affinity and allow for shorter length probes.

A third class, intercalators, are generally flat polycyclic compounds,examples being acridine or lipticine derivatives (see U.S. Pat. No.4,835,263). Intercalating compounds stabilize a duplex by fitting inbetween the bases of the nucleic acid monomers. They can be covalentlyor noncovalently bound. Some minor groove compounds, such as4′,6-diamidino-2-phenylindole (DAPI), also intercalate.

Another group of compounds, capping reagents, are terminally attachedcompounds that favor Watson-Crick duplexes by stacking on the terminalbase pair (Dogan, Z. et al., J. Am. Chem. Soc. 2004, 126, 4762-4763).Such groups include stilbene derivatives (Wu, T. et al., J. Am. Chem.Soc. 1995, 117, 8785-8792) and pyrenylmethylpyrrolindol (Narayanan, S.et al., Nucleic Acids Res. 2004, 32, 2901-2911).

The efficiency of quenching through FRET is extremely sensitive to thedistance between the fluorophore and quencher (R_(FQ)), varying with thereciprocal of R_(FQ) to the sixth power. Maximally efficient quenchingminimizes background fluorescence and improves the sensitivity of the5′-nuclease assay and other hybridization assays in which DLPs are used.Generally, for ease of synthesis and to avoid disruption ofhybridization of the probe to the target sequence, the dye and quencherare attached to the ends of the oligonucleotide. For the 5′-nucleaseassay, the most common configuration is to attach the dye at the 5′-endof the oligonucleotide and the quencher at the 3′-end. DLPs used in the5′-nuclease assay are typically 25 to 30 bases in length. Even with theuse of T_(m) enhancing modifications, such as LNA bases or a minorgroove binder, probe length is still usually 14 to 18 bases. Any methodthat permits placement of the fluorophore and quencher in closerproximity within a probe without destabilizing the duplex formed betweenthe probe and its target nucleic acid will improve quencher efficiencyand enhance the performance of the probe.

BRIEF SUMMARY

This disclosure provides various compositions comprisingoligonucleotides having modifying compounds placed internally within theoligonucleotide sequence between nucleotides. Even though thesemodifying compounds are inserted between adjacent nucleotides (asopposed to being substituted for one of the nucleotides), some of themodified oligonucleotides surprisingly form equally stable, or even morestable, duplexes with their complimentary oligonucleotide sequences ascompared to the stability of the duplex formed between the unmodifiedoligonucleotide and the complimentary oligonucleotide. The modifyinggroups may include a variety of labels, including but not limited tofluorescence quenchers that enable the design of DLPs with very highquenching efficiency. Because the labeling group is not a modified base,the same modifying compound can be inserted into any position within anyoligonucleotide sequence. This disclosure also provides methods forusing and making the oligonucleotide compositions.

The compositions of the present disclosure each comprise anoligonucleotide having the general structure 5′-Y₁-L₁-X-L₂-Y₂-3′, where:

-   -   Y₁ comprises a sequence of one or more DNA and/or RNA        nucleotides, including a first nucleotide N₁ having a 3′        phosphate covalently linked to L₁;    -   Y₂ comprises a sequence of one or more DNA and/or RNA        nucleotides, including a second nucleotide N₂ having a 5′        phosphate covalently linked to L₂;    -   L₁ and L₂ each independently are a direct bond or a C₁-C₇ alkyl,        alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl,        heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl        group;    -   X is

-   -   R₁ is a hydrogen or a C₁-C₈ alkyl; and    -   M is a label.        These oligonucleotides may include any desired number of        nucleotides, but preferably include 10-50 nucleotides, and even        more preferably include 15-35 nucleotides. In some embodiments,        L₁ and L₂ each may be a C₁-C₇ alkyl, and preferably a C₂ alkyl.        The 3′ phosphate that is covalently linked to L₁, and the 5′        phosphate that is covalently linked to L₂, each independently        may be a phosphodiester, a phosphothioate, a phosphodithioate, a        methyl phosphonate, a phosphoramidate, a phosphoramidite or a        phosphotriester.

In some embodiments, Y₁ comprises a sequence of four or more DNA and/orRNA nucleotides, Y₂ comprises a sequence of four or more DNA and/or RNAnucleotides, M is a first quencher, and the oligonucleotide is adaptedto hybridize to a second oligonucleotide having the structure3′-Y₃-Y₄-5′, where Y₃ comprises a sequence of four or more DNA and/orRNA nucleotides, including a third nucleotide N₃, Y₄ comprises asequence of four or more DNA and/or RNA nucleotides, including a fourthnucleotide N₄ that is directly attached to nucleotide N₃. If the firstoligonucleotide hybridizes to the second oligonucleotide, then N₁ basepairs with N₃ and N₂ base pairs with N₄. to form a duplex having a T_(m)that is greater than the T_(m) of a duplex formed between the secondoligonucleotide and a third oligonucleotide having the structure5′-Y₁-Y₂-3′. In such embodiments, the first oligonucleotide may belabeled with a fluorophore. For example, the fluorophore may be attachedto the last nucleotide on the 5′ end of the oligonucleotide, and inpreferred embodiments, Y₁ may comprise a sequence of 8-12 DNA or RNAnucleotides. Compositions comprising the first oligonucleotide also maycomprise the second oligonucleotide.

In some embodiments, Y₁ comprises a sequence of 8-12 DNA and/or RNAnucleotides, such as a sequence of 10 DNA and/or RNA nucleotides, wherethe nucleotide on the 5′ end of Y₁ is labeled with a fluorophore, and Mis a quencher.

In some embodiments, M comprises a fused polycyclic aromatic moiety.

In some embodiments, M is:

where L₃ is a direct bond or a C₁-C₈ alkyl, alkenyl, alkenyl,heteroalkyl, substituted alkyl, cycloalkyl, or alkoxyl, where R₂-R₆ eachindependently are a hydrogen, an alkyl, an alkenyl, a heteroalkyl, asubstituted alkyl, an aryl, a heteroaryl, a substituted aryl, acycloalkyl, an alkylaryl, an alkoxyl, an electron withdrawing group, oran electron donating group, and where one of R₂-R₆ is —N═N—P, and whereP is a fused polycyclic aromatic moiety. Electron withdrawing groups maybe selected from the group consisting of —NO₂, —SO₃ ⁻, —SO₂ ⁻, —CN,—NCS, a ketone, an alkoxyl, an ether, a carboxylic acid and a sulfonyl.Electron donating group is selected from the group consisting of analkoxyl, a heteroalkoxyl, an alkyl, a cycloalkyl, a heteroalkyl, anamino, an alkylamino, or an arylamino.

In some embodiments, M is -L₄-P, where L₄ is an alkyl, an alkynyl, analkenyl, a heteroalkyl, a substituted alkyl, or an alkoxyl group, and Pis a fused polycyclic aromatic moiety. For example, L₄ may be—CH2-O—CH2-CH2-NH— or any other suitable linker.

Various of the oligonucleotides described herein include fusedpolycyclic aromatic moieties P. In some embodiments, P is

where R₇-R₉ each independently are a hydrogen, an alkoxyl, an alkyl, analkylamino, an arylamino, a cycloalkyl, a heteroalkoxyl, a heteroalkyl,or an amino, and R₁₀-R₁₃ each independently are a hydrogen, a nitro, acyano, a carboxylate, a sulfonyl, a sulfamoyl, an alkenyl, an alkynyl,an amino, an aryl, a heteroaryl, a biaryl, a bialkenyl, a bialkynyl, analkoxycarbonyl or a carbamoyl. In preferred embodiments, R₉ is

In some embodiments, P is

where R₁₄-R₁₉ each independently are a hydrogen, an alkyl, aheteroalkyl, an aryl, a heteroaryl, an electron withdrawing group, or afive or six membered ring structure formed from the R1, R2 pair, the R₃,R₄ pair, the R₄, R₅ pair, or the R₅, R₆ pair.

Some of the oligonucleotides disclosed herein include a fluorophore,which may include, but is not limited to, 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX); 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, 2-p-toluidinyl-6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarindye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5 (Cy5.5),3-1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed), a BODIPY™ dye, benzoxaazole, stilbene and pyrene. In someembodiments, the fluorophore may be attached to the 5′ end, such as tothe phosphate at the 5′ end of the oligonucleotide.

Some of the oligonucleotides disclosed herein include more than onequencher, such as a first internal quencher (described above) and asecond quencher. The second quencher may include, but is not limited todabcyl, Eclipse® quencher, BHQ1, BHQ2 and BHQ3, Iowa Black® FQ, IowaBlack® RQ-n1 or Iowa Black® RQ-n2.

This disclosure also provides methods for using and making theoligonucleotide compositions. Methods for use may include methods fordetecting target nucleic acids within a sample. For example, suchmethods may include contacting the sample with an oligonucleotideadapted to hybridize to the target nucleic acid, where theoligonucleotide includes an internal quencher and a fluorophore, andwhere the fluorescence of the fluorophore is reduced by fluorescenceresonance energy transfer to the quencher or by ground state quenchingby the quencher when the oligonucleotide is not hybridized to the secondoligonucleotide, and detecting an increase in fluorescence indicatingthe presence of the second oligonucleotide in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are structures of modified oligonucleotides testedfor their effects on the stability of the duplex formed between themodified oligonucleotide and its complimentary oligonucleotide. Themodified oligonucleotides contain various modification compoundsinserted between, and attached to the 3′ and 5′ phosphates of adjacentnucleotides.

FIG. 2A is an amplification plot that illustrates the relativefluorescence intensity (R_(n)) of an HPRT q-PCR assay of thesubstitution data set for the FQ quencher located 6, 8, 10 and 12positions from the 5′-fluorophore. FIG. 2B is an amplification plot thatillustrates the baseline adjusted fluorescence of results in FIG. 2A(ΔR_(n)).

FIG. 3A is an amplification plot that illustrates the relativefluorescence intensity (R_(n)) of the HPRT q-PCR assay of the insertiondata set for FQ quencher located 6, 8, 10 and 12 positions from the5′-fluorophore. FIG. 3B is an amplification plot that illustratesbaseline adjusted fluorescence of the data in FIG. 3A (ΔR_(n)).

FIG. 4A is an amplification plot that shows the results of the baselineadjusted substitution analogs s10FQ and s6FQ and FIG. 4B is anamplification plot that shows the results for the baseline adjustedinsertion analogs s10FQ and s6FQ where various plasmid copy targetamounts (2×10², 2×10³, 2×10⁴, 2×10⁵, 2×10⁶ and 2×10⁷) were tested.

FIGS. 5A and 5B are amplification plots (baseline comparison (R_(n)) andbaseline adjusted (ΔR_(n)) comparison respectively) that demonstrate theimproved performance of internal FQ (10) by itself or as a dual quenchedprobe compared to an internal BHQ1 (10) dual quenched probe.

FIGS. 6A and 6B are amplification plots (baseline comparison (R_(n)) andbaseline adjusted (ΔR_(n)) respectively) that demonstrate the increasedperformance of internal FQ (iFQ) by itself or as a dual quenchedcontaining probes compared to internal BHQ1 (iBHQ1) dual quenched probeusing a different probe sequence. FIGS. 7A and 7B are amplificationplots (baseline comparison (R_(n)) and baseline adjusted comparison(ΔR_(n)) respectively) using an AB 7900HT instrument comparing a 5′ FAMlabeled dual quencher probes iFQ-3′FQ and iFQ-3′RQ-n1, illustrating theenhanced performance of an internally quenched probe in conjunction witha different 3′ end quencher.

FIGS. 7A and 7B are amplification plots for the fluorescein (emission520 nm) reporter dye probes, wherein the baseline plots are shown inFIG. 7A and baseline normalized plots are shown in FIG. 7B.

FIGS. 8A and 8B are amplification plots (baseline comparison (R_(n)) andbaseline adjusted comparison (ΔR_(n)) respectively) using an AB 7900HTinstrument comparing a 5′ MAX labeled dual quencher probes iFQ-3′FQ andiFQ-3′RQ-n1, illustrating the enhanced performance of an internallyquenched probe in conjunction with a different 3′ end quencher.

FIGS. 9A and 9B are amplification plots (baseline comparison (R_(n)) andbaseline adjusted comparison (ΔR_(n)) respectively) using an iQ5 BioRadinstrument comparing a CY3 labeled dual quencher probes iFQ-3′FQ andiFQ-3′RQ-n1, illustrating the enhanced performance of an internallyquenched probe in conjunction with a different 3′ end quencher.

FIGS. 10A and 10B are amplification plots (baseline comparison (R_(n))and baseline adjusted comparison (ΔR_(n)) respectively) using a LC480Roche instrument comparing a 5′TEX615 labeled dual quencher probesiFQ-3′FQ and iFQ-3′RQ-n1, illustrating the enhanced performance of aninternally quenched probe in conjunction with a different 3′ endquencher.

FIGS. 11A and 11B are amplification plots (baseline comparison (R_(n))and baseline adjusted comparison (ΔR_(n)) respectively) using a LC480Roche instrument comparing a 5′ CY5 labeled dual quencher probesiFQ-3′FQ and iFQ-3′RQ-n1, illustrating the enhanced performance of aninternally quenched probe in conjunction with a different 3′ endquencher.

FIG. 12 is a side by side baseline adjusted amplification plot (ΔR_(n))where various plasmid copy target amounts (2×10², 2×10³, 2×10⁴, 2×10⁵,2×10⁶, 2×10⁷) were tested for CY5 labeled dual quenched probes with theinternal placement of the FQ quencher is between bases 9 and 10, versus11-12, illustrating the ability to vary the positioning with theinternal quencher.

FIGS. 13A, 13B, 13C, 13D and 13E are bar charts showing the ΔT_(m)caused by modifying an oligonucleotide by inserting a modificationcompound between various nucleotides.

FIGS. 14A and 14B are plots showing the dependence of ΔT_(m) on theposition of the insertion within an oligonucleotide for variousmodification compounds.

DETAILED DESCRIPTION

This disclosure provides various compositions comprisingoligonucleotides having modifying compounds placed internally within theoligonucleotide sequence between nucleotides. Some of the modifiedoligonucleotides surprisingly form equally stable, or even more stable,duplexes with their complimentary oligonucleotide sequences as comparedto the stability of the duplex formed between the unmodifiedoligonucleotide and the complimentary oligonucleotide. The mechanism bywhich some modifying compounds confer stability to a duplex is unknown.It is particularly surprising and unexpected that modifications ofadjacent residues within an oligonucleotide with these compounds shouldincrease duplex stability given the close proximity of the two phosphategroups on either side of reagent when in the double helix. The modifyinggroups may include a variety of labels, including but not limited tofluorescence quenchers that enable the design of DLPs with very highquenching efficiency. Because the labeling group is not a modified base,the same modifying compound can be inserted into any position within anyoligonucleotide sequence. This disclosure also provides methods forusing and making the oligonucleotide compositions.

The compositions of the present disclosure each comprise anoligonucleotide having the general structure 5′-Y₁-L₁-X-L₂-Y₂-3′, where:

-   -   Y₁ comprises a sequence of one or more DNA and/or RNA        nucleotides, including a first nucleotide N₁ having a 3′        phosphate covalently linked to L₁;    -   Y₂ comprises a sequence of one or more DNA and/or RNA        nucleotides, including a second nucleotide N₂ having a 5′        phosphate covalently linked to L₂;    -   L₁ and L₂ each independently are a direct bond or a C₁-C₇ alkyl,        alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl,        heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxyl        group;

X is

-   -   R₁ is a hydrogen or a C₁-C₈ alkyl; and    -   M is a label.        FIG. 1 provides a non-exclusive exemplary list of modified        oligonucleotides having a modifying compound inserted between        adjacent nucleotides. These modified oligonucleotides may        include any desired number of nucleotides, but preferably        include 10-50 nucleotides, and even more preferably include        15-35 nucleotides. Moreover, depending on the application, the        labeled oligonucleotide can be DNA, RNA or a chimeric        oligonucleotide containing both DNA and RNA residues. Modified        nucleosides such as LNA bases, 2′-O-methyl RNA and purine and        pyrimidines analogs also may be included within the sequence.        For use as a probe or primer, the length of the oligonucleotide        is typically between 15 and 35 residues. Because the label is        inserted between adjacent residues (as opposed to being a label        attached to a particular nucleotide) the same modifying compound        may be used to label essentially any sequence.

In some embodiments, L₁ and L₂ each may be a C₁-C₇ alkyl, and preferablya C₂ alkyl. An increase in the stability of the duplex formed uponhybridization of the modified oligonucleotides to its target sequencecan be achieved (see Example 1).

The 3′ phosphate that is covalently linked to L₁, and the 5′ phosphatethat is covalently linked to L₂, each independently may be aphosphodiester, a phosphothioate, a phosphodithioate, a methylphosphonate, a phosphoramidate, a phosphoramidite or a phosphotriester.A modified, neutrally charged phosphorous group could be used that wouldconfer even greater stability.

In some embodiments, Y₁ comprises a sequence of four or more DNA and/orRNA nucleotides, Y₂ comprises a sequence of four or more DNA and/or RNAnucleotides, M is a first quencher, and the oligonucleotide is adaptedto hybridize to a second oligonucleotide having the structure3′-Y₃-Y₄-5′, where Y₃ comprises a sequence of four or more DNA and/orRNA nucleotides, including a third nucleotide N₃, Y₄ comprises asequence of four or more DNA and/or RNA nucleotides, including a fourthnucleotide N₄ that is directly attached to nucleotide N₃. If the firstoligonucleotide hybridizes to the second oligonucleotide, then N₁ basepairs with N₃ and N₂ base pairs with N₄. to form a duplex having a T_(m)that is greater than the T_(m) of a duplex formed between the secondoligonucleotide and a third oligonucleotide having the structure5′-Y₁-Y₂-3′. In such embodiments, the first oligonucleotide may belabeled with a fluorophore. For example, the fluorophore may be attachedto the last nucleotide on the 5′ end of the oligonucleotide, and inpreferred embodiments, Y₁ may comprise a sequence of 8-12 DNA or RNAnucleotides for reasons discussed below. Compositions comprising thefirst oligonucleotide also may comprise the second oligonucleotide.

This disclosure also provides optimized positioning of a quencherrelative to a fluorophore in a DLP. In some embodiments, the fluorophoremay be attached to the nucleotide at the 5′-end of the oligonucleotide(e.g., to the 5′ phosphate) and the quencher may be placed internallywithin the sequence between about 8 and 12 bases from the fluorophore,such as about 10 bases from the fluorophore (see Example 3). As such, insome embodiments, Y₁ comprises a sequence of 8-12 DNA and/or RNAnucleotides, such as a sequence of 10 DNA and/or RNA nucleotides, wherethe nucleotide on the 5′ end of Y₁ is labeled with a fluorophore, and Mis a quencher.

In some embodiments, M comprises a fused polycyclic aromatic moiety.

In some embodiments, M is:

where L₃ may be a direct bond or a C₁-C₈ alkyl, alkenyl, alkenyl,heteroalkyl, substituted alkyl, cycloalkyl, or alkoxyl, where R₂-R₆ eachindependently may be a hydrogen, an alkyl, an alkenyl, a heteroalkyl, asubstituted alkyl, an aryl, a heteroaryl, a substituted aryl, acycloalkyl, an alkylaryl, an alkoxyl, a ligand (e.g., amino acids,peptides, antibodies, fluorophores, biotin, enzyme conjugates, vitamins,steroids and other lipids, carbohydrates, digoxigenin and other haptens,etc.), an electron withdrawing group, or an electron donating group, andwhere one of R₂-R₆ is —N═N—P, where P is a fused polycyclic aromaticmoiety. Electron withdrawing groups may be selected from the groupconsisting of —NO₂, —SO₃ ⁻, —SO₂ ⁻, —CN, —NCS, a ketone, an alkoxyl, anether, a carboxylic acid and a sulfonyl. Electron donating groups may beselected from the group consisting of an alkoxyl, a heteroalkoxyl, analkyl, a cycloalkyl, a heteroalkyl, an amino, an alkylamino, or anarylamino.

In some embodiments, M is -L₄-P, where L₄ may be an alkyl, an alkynyl,an alkenyl, a heteroalkyl, a substituted alkyl, or an alkoxyl group, andP is a fused polycyclic aromatic moiety. For example, L₄ may be—CH2-O—CH2-CH2-NH— or any other suitable linker.

Various of the oligonucleotides described herein include fusedpolycyclic aromatic moieties P. In some embodiments, P may be ananthraquinone quencher having the following formula

where R₇-R₉ each independently are a hydrogen, an alkoxyl, an alkyl, analkylamino, an arylamino, a cycloalkyl, a heteroalkoxyl, a heteroalkyl,or an amino, and R₁₀-R₁₃ each independently are a hydrogen, a nitro, acyano, a carboxylate, a sulfonyl, a sulfamoyl, an alkenyl, an alkynyl,an amino, an aryl, a heteroaryl, a biaryl, a bialkenyl, a bialkynyl, analkoxycarbonyl or a carbamoyl. In preferred embodiments, R₉ is

In some embodiments, P may be an azo quencher having the followingformula

where R₁₄-R₁₉ each independently may be a hydrogen, an alkyl, aheteroalkyl, an aryl, a heteroaryl, an electron withdrawing group, anelectron donating group, or a five or six membered ring structure formedfrom the R1, R2 pair, the R₃, R₄ pair, the R₄, R₅ pair, or the R₅, R₆pair, and where R₂₀ preferably is an electron withdrawing group, andmost preferably —NO₂.

Some of the oligonucleotides disclosed herein include a fluorophore,which may include, but is not limited to, 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX); 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, 2-p-toluidinyl-6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarindye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5 (Cy5.5),3-1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed), a BODIPY™ dye, benzoxaazole, stilbene and pyrene. In someembodiments, the fluorophore may be attached to the 5′ end, such as tothe phosphate at the 5′ end of the oligonucleotide.

Some of the oligonucleotides disclosed herein may include more than onequencher, such as a first internal quencher (described above) and asecond quencher. The second quencher may be placed internally or placedat a terminal end of the oligonucleotide. The second quencher mayinclude, but is not limited to, an azo quencher and an anthraquinonequencher, although any quencher may be used. Examples of azo quenchersinclude, but are not limited to, the azo quencher shown above, dabcyl,Eclipse® quencher, BHQ1, BHQ2 and BHQ3. Examples of anthraquinonequenchers include, but are not limited to, the anthraquinone quenchershown above, Iowa Black® FQ, Iowa Black® RQ-n1 or Iowa Black® RQ-n2(see, e.g., Laikhter et al., U.S. Patent App. 2004/0110308). Attachmentof multiple quenchers to a probe not only can enhance quenchingefficiency but also can provide effective quenching of variousfluorophores that fluoresce over a broad spectral range (see Example 7).

The compositions of the present disclosure may be used in various assaysfor detecting target nucleic acids within a sample. Such methods mayinclude contacting the sample with an oligonucleotide adapted tohybridize to the target nucleic acid, where the oligonucleotide includesan internal quencher and a fluorophore, and where the fluorescence ofthe fluorophore is reduced by fluorescence resonance energy transfer tothe quencher and/or by ground state quenching by the quencher when theoligonucleotide is not hybridized to the second oligonucleotide, anddetecting an increase in fluorescence indicating the presence of thesecond oligonucleotide in the sample. In some assays, such as the5′-nuclease hydrolysis assay, the increase in fluorescence arises fromcleavage of the labeled oligonucleotide. In some assays, theoligonucleotide forms a random-coil conformation when theoligonucleotide is unhybridized, such that the fluorescence of thefluorophore is reduced. In some assays, the oligonucleotide comprises aself-complimentary sequence, and the quencher and fluorophore areattached to the oligonucleotide such that the fluorescence of thefluorophore is quenched by the quencher when the nucleic acid polymerundergoes intramolecular base pairing. These assays have manyapplications, including, but not limited to, monitoring PCR reactions,where synthesis of the PCR product results in an increase influorescence.

Function of dual-labeled probes in the 5′-nuclease hydrolysis assayrequires that the fluorophore be effectively quenched by the quencherand also requires that the chemical modifiers employed (dye andquencher) do not interfere with nuclease cleavage. If cleavage isprevented or rendered inefficient by the presence of the chemicalmodifications, then fluorophore and quencher remain linked during PCRcycles and no detectable signal is generated. The chemical compositionsdisclosed herein function to efficiently quench the fluorophore and arecompatible with probe hydrolysis using 5′-nuclease positive DNApolymerases, like Taq DNA polymerase. The best results are obtained whenthe internal quencher is positioned as close to the fluorophore aspossible (to maximize quenching) yet still permits efficient cleavage ofthe nucleic acid bases between the dye and quencher (maximize theresulting fluorescent signal). Many fluorescent quenching groups can beplaced internally and achieve efficient quenching, however many if notmost of these chemical groups interfere with probe cleavage, especiallywhen the distance between fluorophore and quencher is less than 12nucleotides. This principle is demonstrated in Example 3, where certainquenchers, such as BHQ-1, can achieve very efficient quenching of a5′-fluorophore, such as Fluorescein (6-FAM), yet the actual magnitude ofthe fluorescent signal generated during real-time PCR is small,compromising the actual performance and sensitivity of the assay. Incontrast, similar internal placement of the quenchers provided herein,are fully compatible with probe hydrolysis and final functional signalgeneration is large.

Traditional DLPs having a 5′-fluorophore and 3′-quencher perform poorlywhen probe length nears or exceeds 30 nucleotides (nt), becausequenching efficiency drops to the point that the probe remainsrelatively bright even in the quenched state. The present disclosureprovides probes of 30 nt length, 35 nt length, or longer as needed forthe precise application, having internal quenchers substantially closerto the fluorophore. Quenching in the compositions of this disclosureremains highly efficient as the internal quencher can be inserted thesame distance from the fluorophore regardless of probe length. Thus highquality, efficiently quenched probes of an expanded potential lengthrange are possible, which may be of particular importance when workingwith nucleic acids which are very AT rich. Sequences that are AT-richhave lower melting temperatures and longer probes must be utilized tofunction in the temperature ranges typically needed for PCR.

The compounds of this disclosure can also be utilized in molecularbeacon assays. Molecular beacon assays contain probes that containterminal 3′ and 5′ ends that self-hybridize to form a stem-loopstructure. One end typically contains a terminal fluorophore group andthe other end contains a terminal quencher group. When the probehybridizes to the target the quencher is no longer near the fluorophoreand the signal increases. Typically the hybridizing portions of theprobes are 4-7 base pairs long. Beacon probes that contain insertions asdescribed herein, preferably within the hybridized portion of thebeacon, and more preferably within 1-2 bases from the terminal end, thestability is increased. Therefore shorter hybridizing regions can beused to generate the same performance as a conventional beacon.

A wide variety of reactive fluorescent reporter dyes are known in theliterature and can be used in the compositions of this disclosure, solong as they are quenched by the precise quencher group or combinationof quencher groups employed. The precise fluorophore/quencher pairemployed in a dual-labeled probe is usually carefully chosen from arelatively small set of pairings that work well together based upon theemission wavelength of the fluorophore and the absorption wavelength ofthe quencher. Placement of the quencher in an internal position, asprovided here, closer to the fluorophore than is achievable with anend-labeled probe permits efficient quenching even offluorophore/quencher pairs that usually do not work well together,expanding the utility of the quencher by enabling its use with a widerrange of fluorophores. Attachment of a second quencher to the probe caneven further expand the useful spectral range.

The oligonucleotide probes provided herein may incorporate one or morefluorophores. The fluorophores can be attached internally or at the 5′-or 3′-end. Typically, the fluorophore is an aromatic or heteroaromaticcompound and can be a pyrene, anthracene, naphthalene, acridine,stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine,carbocyanine, salicylate, anthranilate, coumarin, fluoroscein, rhodamineor other like compound. Suitable fluorescent reporters include xanthenedyes, such as fluorescein or rhodamine dyes, including6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX). Suitable fluorescent reporters also include the naphthylaminedyes that have an amino group in the alpha or beta position. Forexample, naphthylamino compounds include1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-toluidinyl-6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Otherfluorescent reporter dyes include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such asindodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),indodicarbocyanine 5.5 (Cy5.5),3-1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2 (or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4 (or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed); BODIPY™ dyes; benzoxaazoles; stilbenes; pyrenes; and the like. SeeHaugland, “Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals” for further fluorophore examples.

Reagents for incorporation the modification compounds of the presentdisclosure into oligonucleotides may have the following generalstructure:

where R₁₃ is a protecting group on the oxygen atom, most commonly atrityl group, and preferably a dimethoxytrityl group, and R₂₂ is aphosphoramidite, a phosphate group, or a hydrogen phosphate used tocouple the reagent to the growing oligonucleotide chain duringsynthesis. A phosphoramidite is preferred. AN,N-diisopropyl-β-cyanoethyl phosphoramidite is the most preferredreactive group.

As used herein, the terms “nucleic acid” and “oligonucleotide,” as usedherein, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N glycoside of a purine or pyrimidine base.There is no intended distinction in length between the terms “nucleicacid”, “oligonucleotide”, “oligomer” or “oligo”, and these terms will beused interchangeably. These terms refer only to the primary structure ofthe molecule. Thus, these terms include double- and single-stranded DNA,as well as double- and single-stranded RNA. An oligonucleotide also cancomprise nucleotide analogs in which the base, sugar, or phosphatebackbone is modified as well as non-purine or non-pyrimidine nucleotideanalogs oligonucleotides, which may comprise naturally occurringnucleosides or chemically modified nucleosides. In some embodiments, thecompounds comprise modified sugar moieties, modified internucleosidelinkages, or modified nucleobase moieties.

The term “base” as used herein includes purines, pyrimidines andnon-natural bases and modifications well-known in the art. Purinesinclude adenine, guanine and xanthine and modified purines such as8-oxo-N⁶-methyladenine and 7-deazaxanthine. Pyrimidines include thymine,uracil and cytosine and their analogs such as 5-methylcytosine and4,4-ethanocytosine. Non-natural bases include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,nitroindole, and 2,6-diaminopurine.

The term “base” is sometimes used interchangeably with “monomer”, and inthis context it refers to a single nucleic acid or oligomer unit in anucleic acid chain.

The term “probe” as used herein refers to nucleic acid oligonucleotidesthat produce a detectable response upon interaction with a target. Theprobes include at least one detectable moiety, a pair of moieties thatform an energy transfer pair detectable upon some change of state of theprobe in response to its interaction with a binding partner, or morethan two moieties such as a fluorophore and more than one quencher.

The term “primer,” as used herein, refers to an oligonucleotide capableof acting as a point of initiation of DNA synthesis under suitableconditions. Such conditions include those in which synthesis of a primerextension product complementary to a nucleic acid strand is induced inthe presence of four different nucleoside triphosphates and an agent forextension (e.g., a DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature. A primer is preferablya single-stranded DNA. The appropriate length of a primer depends on theintended use of the primer but typically ranges from 6 to 50nucleotides, preferably from 15-35 nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template. A primer need not reflect the exactsequence of the template nucleic acid, but must be sufficientlycomplementary to hybridize with the template. The design of suitableprimers for the amplification of a given target sequence is well knownin the art and described in the literature cited herein. Primers canincorporate additional features which allow for the detection orimmobilization of the primer but do not alter the basic property of theprimer, that of acting as a point of initiation of DNA synthesis. Forexample, primers may contain an additional nucleic acid sequence at the5′ end which does not hybridize to the target nucleic acid, but whichfacilitates cloning or detection of the amplified product. The region ofthe primer which is sufficiently complementary to the template tohybridize is referred to herein as the hybridizing region.

The term “hybridization,” as used herein, refers to the formation of aduplex structure by two single-stranded nucleic acids due tocomplementary base pairing. Hybridization can occur between fullycomplementary nucleic acid strands or between “substantiallycomplementary” nucleic acid strands that contain minor regions ofmismatch. Conditions under which hybridization of fully complementarynucleic acid strands is strongly preferred are referred to as “stringenthybridization conditions” or “sequence-specific hybridizationconditions”. Stable duplexes of substantially complementary sequencescan be achieved under less stringent hybridization conditions; thedegree of mismatch tolerated can be controlled by suitable adjustment ofthe hybridization conditions. Those skilled in the art of nucleic acidtechnology can determine duplex stability empirically considering anumber of variables including, for example, the length and base paircomposition of the oligonucleotides, ionic strength, and incidence ofmismatched base pairs, following the guidance provided by the art (see,e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991,Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzyet al., 2008, Biochemistry, 47: 5336-5353, which are incorporated hereinby reference).

The term “amplification reaction” refers to any chemical reaction,including an enzymatic reaction, which results in increased copies of atemplate nucleic acid sequence or results in transcription of a templatenucleic acid. Amplification reactions include reverse transcription, thepolymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat.Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)), and the ligase chain reaction(LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary“amplification reactions conditions” or “amplification conditions”typically comprise either two or three step cycles. Two step cycles havea high temperature denaturation step followed by ahybridization/elongation (or ligation) step. Three step cycles comprisea denaturation step followed by a hybridization step followed by aseparate elongation or ligation step.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the improved stability of probes containingmodifications of the present disclosure compared to a selection of othercompounds.

Oligonucleotide synthesis and purification. DNA oligonucleotides weresynthesized using solid phase phosphoramidite chemistry, deprotected anddesalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.)according to routine techniques (Caruthers et al., Methods Enzymol 1992,211:3-20). The oligomers were purified using reversed-phase highperformance liquid chromatography (RP-HPLC). The purity of each oligomerwas determined by capillary electrophoresis (CE) carried out on aBeckman P/ACE MDQ system (Beckman Coulter, Inc., Fullerton, Calif.). Allsingle strand oligomers were at least 90% pure. Electrospray-ionizationliquid chromatography mass spectroscopy (ESI-LCMS) of theoligonucleotides was conducted using an Oligo HTCS system (Novatia,Princeton, N.J.), which consisted of ThermoFinnigan TSQ7000, Xcaliburdata system, ProMass data processing software and Paradigm MS4™ HPLC(Michrom BioResources, Auburn, Calif.). Protocols recommended bymanufacturers were followed. Experimental molar masses for all singlestrand oligomers were within 1.5 g/mol of expected molar mass. Theseresults confirm identity of the oligomers.

Preparation of DNA samples. Melting experiments were carried out inbuffer containing 3.87 mM NaH₂PO₄, 6.13 mM Na₂HPO₄, 1 mM Na₂EDTA and1000 mM NaCl. 1 M NaOH was used to titrate each solution to pH 7.0.Total sodium concentrations were estimated to be 1020 mM. The DNAsamples were thoroughly dialyzed against melting buffer in a 28-WellMicrodialysis System (Life Technologies, Carlsbad, Calif.) following themanufacturer's recommended protocol. Concentrations of DNA oligomerswere estimated from the samples' UV absorbance at 260 nm in aspectrophotometer (Beckman Coulter, Inc., Fullerton, Calif.), usingextinction coefficients for each oligonucleotide that were estimatedusing the nearest neighbor model for calculating extinctioncoefficients. (See, Warshaw et al., J. Mol. Biol. 1966, 20:29-38).

Internal modifications studied. FIGS. 1A-1C show the structures ofmodified portions of the various modified oligonucleotides studied inthis example and in Example 2. The FQ (Integrated DNA Technologies,Inc., sometimes referred to as “iFQ” in this application), wasintroduced into oligonucleotides using phosphoramidite reagents at thetime of synthesis. See Example 10 for synthesis of the phosphoramidite.In the first series of duplexes, the iFQ group was placed as aninsertion between bases in the duplex so that a 10 base top strandannealed to a 10 base bottom strand and the iFQ group was not aligned toa base. Additionally, 10-mer oligonucleotides with C3 spacer insertionswere also synthesized and studied. The C3 spacer represents the controlwherein a linear insertion of a phosphate group plus propanediol isplaced between bases, which is similar to the iFQ insertions withouthaving the nathylene-azo ring structures present. Extinctioncoefficients at 260 nm of iFQ was estimated to be 13340; the C3 spacerdoes not contribute to UV absorbance. Two 20 base and two 25 baseduplexes of similar design were also studied. A second set of 10 baseduplexes was studied where the iFQ group was placed as a substitutionsuch that a 10 base top strand (5 bases-iFQ-5 bases) was annealed to an11 base bottom strand so that the iFQ group functioned as a substitutionor replacement for a base. Four duplexes of this design were tested, onecomprising each of the 4 bases (AGCT) to pair with the iFQ group.

Measurement of melting curves. Oligomer concentrations were measured atleast twice for each sample. If the estimated concentrations for anysample differed more than 4%, the results were discarded and newabsorbance measurements were performed. To prepare oligonucleotideduplexes, complementary DNA oligomers were mixed in 1:1 molar ratio,heated to 367 K (i.e., 94° C.) and slowly cooled to an ambienttemperature. Each solution of duplex DNA was diluted with melting bufferto a total DNA concentration (C_(T)) of 2 μM.

Melting experiments were conducted on a single beam Beckman DU 650spectrophotometer (Beckman-Coulter) with a Micro T_(m) Analysisaccessory, a Beckman High Performance Peltier Controller (to regulatethe temperature), and 1 cm path-length cuvettes. Melt data were recordedusing a PC interfaced to the spectrophotometer. UV-absorbance values at268 nm wavelength were measured at 0.1 degree increments in thetemperature range from 383 to 368 K (i.e., 10-95° C.). Both heating(i.e., denaturation) and cooling (i.e., renaturation) transition curveswere recorded in each sample at a controlled rate of temperature change(24.9±0.3° C. per hour). Sample temperatures were collected from theinternal probe located inside the Peltier holder, and recorded with eachsample's UV-absorbance data. Melting profiles were also recorded forsamples of buffer alone (no oligonucleotide), and these blank profileswere digitally subtracted from melting curves of the DNA samples. Tominimize systematic errors, at least two melting curves were collectedfor each sample in different cuvettes and in different positions withinthe Peltier holder.

Determination of melting temperatures. To determine each sample'smelting temperature, the melting profiles were analyzed using methodsthat have been previously described (see, Doktycz et al., Biopolymers1992, 32:849-864; Owczarzy et al., Biopolymers 1997, 44:217-239;Owczarzy R., Biophys. Chem. 2005, 117: 207-215). Briefly, theexperimental data for each sample was smoothed, using a digital filter,to obtain a plot of the sample's UV-absorbance as a function of itstemperature. The fraction of single-stranded oligonucleotide molecules,θ, was then calculated from that plot. The melting temperature or T_(m)of a sample was defined as the temperature where θ=0.5.

Table 1 lists the sequences tested, the internal quenchers used, and theresulting melting temperatures.

TABLE 1 Melting Temperatures for nucleic acids containing internal quencher moieties, where iFQ = internal FQ azo quencher,   and iSpC3 =internal C3 spacer. SEQ ID NO. Duplex Sequence N T_(m)(C.) ΔT_(m)(C.) 15′-ATCGTTGCTA-3′ 10 43.85 0.0 2 3′-TAGCAACGAT-5′ 10 35′-ATC/iFQ/GTTGCTA-3′ 10 48.05 4.2 2 3′-TAGCAACGAT-5′ 10 45′-ATCG/iFQ/TTGCTA-3′ 10 48.55 4.7 2 3′-TAGCAACGAT-5′ 10 55′-ATCGT/iFQ/TGCTA-3′ 10 46.35 2.5 2 3′-TAGCAACGAT-5′ 10 65′-CTTGGATCGTTGCTAGTAGG-3′ 20 69.55 0.0 7 3′-GAACCTAGCAACGATCATCC-5′ 208 5′-CTTGGATCGT/iFQ/TGCTAGTAGG-3′ 20 71.35 1.8 73′-GAACCTAGCAACGATCATCC-5′ 20 9 5′-CACTTGGATCGTTGCTAGTAGGGTC-3′ 25 76.150.0 10 3′-GTGAACCTAGCAACGATCATCCCAG-5′ 25 115′-CACTTGGATC/iFQ/GTTGCTAGTAGGGTC-3′ 25 77.05 0.9 103′-GTGAACCTAGCAACGATCATCCCAG-5′ 25 12 5′-ATC/iSpC3/GTTGCTA-3′ 10 36.35−7.5 2 3′-TAGCAACGAT-5′ 10 13 5′-ATCG/iSpC3/TTGCTA-3′ 10 36.55 −7.3 23′-TAGCAACGAT-5′ 10 14 5′-ATCGT/iSpC3/TGCTA-3′ 10 32.55 −11.3 23′-TAGCAACGAT-5′ 10 5 5′-ATCGT/iFQ/TGCTA-3′ 10 47.35 3.5 153′-TAGCA/iSpC3/ACGAT-5′ 10 5 5′-ATCGT/iFQ/TGCTA-3′ 10 42.24 −1.6 163′-TAGCAAACGAT-5′ 11 5 5′-ATCGT/iFQ/TGCTA-3′ 10 45.27 1.4 173′-TAGCACACGAT-5′ 11 5 5′-ATCGT/iFQ/TGCTA-3′ 10 40.44 −3.4 183′-TAGCAGACGAT-5′ 11 5 5′-ATCGT/iFQ/TGCTA-3′ 10 45.27 1.4 193′-TAGCATACGAT-5′ 11

Three different insertion placement sites were studied using a 10-meroligonucleotide scaffold. Use of the shorter sequences most clearlydemonstrates the potential effects on T_(m) and testing differentplacement sites illustrate that the T_(m) effects can be sequencecontext dependent. The relative ΔT_(m) shifts for the modified vs.unmodified 10mer sequences were averaged and are summarized in Table 2below.

TABLE 2 Average ΔT_(m) shifts for three 10 mer sequences with internalmodifiers Modifier iFQ (insertion) iFQ (substitution) iSpC3 ΔT_(m) +3.8°C. −0.6° C. −8.7° C.

As Tables 1 and 2 illustrate, disrupting a DNA sequence with amodification that is small and offers no steric hindrance (orstabilization) like a propanediol group (C3 spacer) has a significantnegative impact on the T_(m) of a duplex (ΔT_(m) of −8.7° C.). Incontrast, the napthylene-azo-class quencher studied (iFQ) significantlystabilized the duplex compared with the iC3 control. A greater degree ofstabilization was seen when the iFQ was placed as an insertion (ΔT_(m)of +12.5° C. relative to the iSpC3) than when the iFQ was placed as abase substitution (ΔT_(m) of +8.1° C. relative to the iSpC3).Unexpectedly, use of the iFQ group as an insertion between basesstabilized the duplex compared to the unmodified parent duplex (ΔT_(m)of +3.8° C. relative to the unmodified duplex), while base substitutionresulted in slight destabilization (ΔT_(m) of −0.6° C. relative to theunmodified duplex).

Therefore internal incorporation of the napthylene-azo group within aDNA duplex stabilizes the duplex when placed as an insertion betweenbases.

Certain anthraquinone groups can stabilize a duplex when placed on theends (J. Am. Chem. Soc., 131:12671-12681, 2009); however this effect hasnot been described for internal placement or using napthylene-azocompounds. Therefore the use of internal napthylene-azo-class quencherswould be preferred to maintain duplex stability.

EXAMPLE 2

The following example compares 10 base pair sets of duplexes withvarying modification insertions placed at varying location along the10-mer oligonucleotide. The structures of the modified portions of thevarious modified oligonucleotides are illustrated in FIGS. 1A-1C. Asnoted earlier, the synthesis of FQ phosphoramidite is described inExample 10. The structure “IB 1.1” is synthesized in the same manner asFQ except an aminoanthraquinone reagent is used instead of4-nitro-1-napthylamine. The IB RQ quenchers are anthraquinone-basedcompounds (U.S. Pat. Application 2004/0110308) which are commonly usedwith red wavelength fluorescent dyes.

The preparation of the DNA samples, the measurement of melting curvesand determination of melting temperatures were performed as inExample 1. Table 3 lists the resulting Tm data for each duplex studied,as well as the ΔT_(m) relative to the duplex formed with the unmodifiedoligonucleotide.

TABLE 3 SEQ ID No. Sequence T_(m) ΔT_(m) 1 5′ ATCGTTGCTA 43.9 — 2 3′TAGCAACGAT 20 5′ ATC/GTTGCTA N-MDA “/” 31.0 −12.9 2 3′ TAG CAACGAT 21 5′ATC/GTTGCTA C2 “/” 37.8  −6.1 2 3′ TAG CAACGAT 22 5′ATC/GTTGCTA 2,2 DMP “/” 34.9  −9.0 2 3′ TAG CAACGAT 12 5′ATC/GTTGCTA iSpC3 “/” 36.3  −7.6 2 3′ TAG CAACGAT 23 5′ATC/GTTGCTA C4 “/” 30.1 −13.8 2 3′ TAG CAACGAT 24 5′ ATC/GTTGCTA C5 “/”27.3 −16.6 2 3′ TAG CAACGAT 25 5′ ATC/GTTGCTA C6 “/” 26.1 −17.8 2 3′TAG CAACGAT 26 5′ ATC/GTTGCTA C7 “/” 24.7 −19.1 2 3′ TAG CAACGAT 27 5′ATC/GTTGCTA iSpS9 “/” 28.5 −15.4 2 3′ TAG CAACGAT 28 5′ATC/GTTGCTA idSp “/” 33.5 −10.4 2 3′ TAG CAACGAT 3 5′ATC/GTTGCTA iFQ “/” 48.0  +4.1 2 3′ TAG CAACGAT 29 5′ATC/GTTGCTA iBHQ2 “/” 45.0  +1.1 2 3′ TAG CAACGAT 30 5′ATC/GTTGCTA iRQ-n1 “/” 38.7  −5.2 2 3′ TAG CAACGAT 31 5′ATC/GTTGCTA iRQ-n2 “/” 46.0  +2.1 2 3′ TAG CAACGAT 32 5′ATC/GTTGCTA iEc “/” 40.1  −3.8 2 3′ TAG CAACGAT 33 5′ATC/GTTGCTA IB 1.1 “/” 47.6  +3.7 2 3′ TAG CAACGAT 34 5′ATC/GTTGCTA NPDA “/” 29.3 −14.6 2 3′ TAG CAACGAT 35 5′ATCG/TTGCTA N-MDA “/” 34.1  −9.8 2 3′ TAGC AACGAT 36 5′ATCG/TTGCTA C2 “/” 38.6  −5.3 2 3′ TAGC AACGAT 37 5′ATCG/TTGCTA 2,2 DMP “/” 35.5  −8.4 2 3′ TAGC AACGAT 13 5′ATCG/TTGCTA iSpC3 “/” 36.6  −7.3 2 3′ TAGC AACGAT 38 5′ATCG/TTGCTA C4 “/” 32.0 −11.9 2 3′ TAGC AACGAT 39 5′ ATCG/TTGCTA C5 “/”28.1 −15.8 2 3′ TAGC AACGAT 40 5′ ATCG/TTGCTA C6 “/” 26.5 −17.4 2 3′TAGC AACGAT 41 5′ ATCG/TTGCTA C7 “/” 24.7 −19.2 2 3′ TAGC AACGAT 42 5′ATCG/TTGCTA iSpS9 “/” 29.1 −14.8 2 3′ TAGC AACGAT 43 5′ATCG/TTGCTA idSp “/” 34.1  −9.8 2 3′ TAGC AACGAT 4 5′ATCG/TTGCTA iFQ “/” 48.6  +4.7 2 3′ TAGC AACGAT 44 5′ATCG/TTGCTA iBHQ2 “/” 43.3  −0.6 2 3′ TAGC AACGAT 45 5′ATCG/TTGCTA iRQ-n1 “/” 34.7  −9.2 2 3′ TAGC AACGAT 46 5′ATCG/TTGCTA iRQ-n2 “/” 44.7  +0.8 2 3′ TAGC AACGAT 47 5′ATCG/TTGCTA iEc “/” 35.7  −8.2 2 3′ TAGC AACGAT 48 5′ATCG/TTGCTA IB 1.1 “/” 45.9  +2.1 2 3′ TAGC AACGAT 49 5′ATCG/TTGCTA NPDA “/” 30.0 −13.9 2 3′ TAGC AACGAT 50 5′ATCGT/TGCTA C2 “/” 34.0  −9.9 2 3′ TAGCA ACGAT 51 5′ATCGT/TGCTA 2,2 DMP “/” 33.1 −10.8 2 3′ TAGCA ACGAT 14 5′ATCGT/TGCTA iSpC3 “/” 32.6 −11.3 2 3′ TAGCA ACGAT 52 5′ATCGT/TGCTA C4 “/” 26.4 −17.5 2 3′ TAGCA ACGAT 53 5′ ATCGT/TGCTA C5 “/”23.3 −20.6 2 3′ TAGCA ACGAT 54 5′ ATCGT/TGCTA C6 “/” 21.9 −22.0 2 3′TAGCA ACGAT 55 5′ ATCGT/TGCTA C7 “/” 20.4 −23.5 2 3′ TAGCA ACGAT 56 5′ATCGT/TGCTA iSpS9 “/” 24.4 −19.5 2 3′ TAGCA ACGAT 57 5′ATCGT/TGCTA idSp “/” 31.8 −12.1 2 3′ TAGCA ACGAT 5 5′ATCGT/TGCTA iFQ “/” 46.3  +2.4 2 3′ TAGCS ACGAT 58 5′ATCGT/TGCTA iBHQ2 “/” 41.5  −2.4 2 3′ TAGCA ACGAT 59 5′ATCGT/TGCTA iRQ-n1 “/” 32.1 −11.8 2 3′ TAGCA ACGAT 60 5′ATCGT/TGCTA iRQ-n2 “/” 42.6  −1.3 2 3′ TAGCA ACGAT 61 5′ATCGT/TGCTA iEc “/” 33.1 −10.8 2 3′ TAGCA ACGAT 62 5′ATCGT/TGCTA IB 1.1 “/” 44.7  +0.8 2 3′ TAGCA ACGAT 63 5′ATCGT/TGCTA NPDA “/” 26.8 −17.1 2 3′ TAGCA ACGAT 64 5′A/TCGTTGCTA N-MDA “/” 40.9  −3.0 2 3′ T AGCAACGAT 65 5′A/TCGTTGCTA C2 “/” 43.5  −0.4 2 3′ T AGCAACGAT 66 5′A/TCGTTGCTA 2,2 DMP “/” 43.4  −0.4 2 3′ T AGCAACGAT 67 5′A/TCGTTGCTA iSpC3 “/” 44.6  +0.7 2 3′ T AGCAACGAT 68 5′A/TCGTTGCTA C4 “/” 43.3  −0.6 2 3′ T AGCAACGAT 69 5′ A/TCGTTGCTA C5 “/”41.5  −2.4 2 3′ T AGCAACGAT 70 5′ A/TCGTTGCTA C6 “/” 41.6  −2.3 2 3′T AGCAACGAT 71 5′ A/TCGTTGCTA C7 “/” 43.2  −0.7 2 3′ T AGCAACGAT 72 5′A/TCGTTGCTA iSpS9 “/” 41.5  −2.4 2 3′ T AGCAACGAT 73 5′A/TCGTTGCTA idSp “/” 43.0  −0.9 2 3′ T AGCAACGAT 74 5′A/TCGTTGCTA iFQ “/” 51.8  +7.9 2 3′ T AGCAACGAT 75 5′A/TCGTTGCTA iBHQ2 “/” 48.9  +5.0 2 3′ T AGCAACGAT 76 5′A/TCGTTGCTA iRQ-n1 “/” 48.2  +4.3 2 3′ T AGCAACGAT 77 5′A/TCGTTGCTA iRQ-n2 “/” 49.1  +5.2 2 3′ T AGCAACGAT 78 5′A/TCGTTGCTA iEc “/” 44.5  +0.6 2 3′ T AGCAACGAT 79 5′A/TCGTTGCTA IB 1.1 “/” 51.2  +7.3 2 3′ T AGCAACGAT 80 5′A/TCGTTGCTA NPDA “/” 41.0  −2.9 2 3′ T AGCAACGAT 81 5′ATCGTTGCT/A C2 “/” 44.2  +0.3 2 3′ TAGCAACGA T 82 5′ATCGTTGCT/A 2,2 DMP “/” 43.8  −0.1 2 3′ TAGCAACGA T 83 5′ATCGTTGCT/A iSpC3 “/” 44.5  +0.6 2 3′ TAGCAACGA T 84 5′ATCGTTGCT/A C4 “/” 43.4  −0.5 2 3′ TAGCAACGA T 85 5′ ATCGTTGCT/A C5 “/”42.8  −1.0 2 3′ TAGCAACGA T 86 5′ ATCGTTGCT/A C6 “/” 43.3  −0.6 2 3′TAGCAACGA T 87 5′ ATCGTTGCT/A C7 “/” 40.9  −3.0 2 3′ TAGCAACGA T 88 5′ATCGTTGCT/A iSpS9 “/” 44.2  +0.3 2 3′ TAGCAACGA T 89 5′ATCGTTGCT/A idSp “/” 44.7  +0.8 2 3′ TAGCAACGA T 90 5′ATCGTTGCT/A iFQ “/” 50.3  +6.4 2 3′ TAGCAACGA T 91 5′ATCGTTGCT/A iBHQ2 “/” 50.7  +6.8 2 3′ TAGCAACGA T 92 5′ATCGTTGCT/A iRQ-n1 “/” 48.6  +4.7 2 3′ TAGCAACGA T 93 5′ATCGTTGCT/A iRQ-n2 “/” 47.5  +3.6 2 3′ TAGCAACGA T 94 5′ATCGTTGCT/A iEc “/” 45.8  +1.9 2 3′ TAGCAACGA T 95 5′ATCGTTGCT/A IB 1.1 “/” 50.9  +7.0 2 3′ TAGCAACGA T 96 5′ATCGTTGCT/A NPDA “/” 43.9  +0.0 2 3′ TAGCAACGA T

FIGS. 13A, 13B, 13C, 13D and 13E are bar charts each showing the ΔT_(m)caused by each of the modifications at a particular location within thenucleotide. FIGS. 14A and 14B are plots showing the dependence of ΔT_(m)on the position of the insertion within an oligonucleotide for each ofthe various modification compounds. In general, the insertion of amodification nearer the end of an oligonucleotide is less destabilizingthan when it is inserted towards the middle of the oligonucleotide. Thespacer modifications were all destabilizing, with the degree ofdestabilization increasing as the size of the spacer increased.

The FQ modification had a positive effect on stability whether it wasinserted near an end or in the middle of the duplex. The IB1.1 hadnearly the same stability profile as FQ. The iRQ-n2 modification wasalso positive or negligible in its effect on duplex stability.

EXAMPLE 3

This example details the predictive modeling for oligonucleotidescontaining an internal quencher.

The thermodynamic impact of internal FQ azo quencher (iFQ) modificationwas determined from the difference between modified and native duplexDNAs. Melting experiments were conducted at 2 μM DNA concentration(C_(t)) and in 1M Na⁺ buffer. Transition enthalpies (ΔH°) and entropies(ΔS°) where obtain from fits to individual melting profiles (Petersheim,M.& Turner, D. H. (1983) Biochemistry, 22, 256-263). The equilibriumconstant K_(a) was calculated from the fraction of broken base pairs (θ)at each temperature,

$\begin{matrix}{K_{a} = \frac{2\left( {1 - \theta} \right)}{\theta^{2}C_{t}}} & (1)\end{matrix}$

The graphs of −ln K_(a) vs. 1/T were least-square fit to straight linesand thermodynamic parameters were calculated from the slope and theintercept,

$\begin{matrix}{{{- \ln}\; K_{a}} = {\frac{\Delta\; H^{o}}{RT} - \frac{\Delta\; S^{o}}{R}}} & (2)\end{matrix}$

The fits were limited to the range of θ from 0.15 to 0.85, where θ andK_(a) are the most accurate. The symbol R is the ideal gas constant(1.9865 cal/(mol*K)). Reported values of ΔH°, and ΔS° are averages fromat least four heating and cooling melting profiles. This thermodynamicanalysis assumes that the transition enthalpies and entropies aretemperature-independent and melting transitions proceed in two-statemanner. The results are summarized in Table 4. Average thermodynamiceffect of inserted FQ azo quencher could be estimated from the followingrelationships,ΔH° (modified oligo)=ΔH° (native oligo)−1 272 cal/molΔS° (modified oligo)=ΔS° (native oligo)−1.44 cal/(mol.K)When these equations were employed to predict melting temperatures of 20and 25 base pair duplexes (Table 5), the average error of predictionswas 0.7° C.

TABLE 4 Thermodynamic effects of internal FQ modifications SEQ IDDNA sequence ΔH° ΔΔH° ΔS° ΔΔS° No. (5′ to 3′)^(a) cal/mol cal/molcal/(mol.K) cal/(mol.K) 1 ATCGTTGCTA −68012 −185.62 3 ATC/iFQ/GTTGCTA−71827 −3815 −194.73 −9.12 4 ATCG/iFQ/TTGCTA −69783 −1771 −188.00 −2.385 ATCGT/iFQ/TGCTA −66241  1771 −178.45 7.17 Average effect −1272 −1.44^(a)Complementary DNA strand was 5′-TAGCAACGAT-3′. Calculations weredone using non-rounded values.

TABLE 5Accuracy of T_(m) predictions for two duplex DNAs containing internal FQ quencher that were not used to derive equations (1) and (2). Error ofSEQ Exper. Predicted T_(m) ID T_(m) T_(m) prediction No.DNA sequence (5′ to 3′)^(a) N_(bp) (° C.) (° C.) (° C.)  8CTTGGATCGT/iFQ/TGCTAGTAGG 20 71.3 71.6 0.3 11CACTTGGATC/iFQ/GTTGCTAGTAGGGTC 25 77.1 78.2 1.1 ^(a)Melting temperatureswere calculated using the nearest-neighbor model (SantaLucia, J., Jr.(1998) Proc. Natl. Acad. Sci. USA, 95, 1460-1465) and equations (1) and(2).

The low error of prediction demonstrates the consistency andpredictability of the effect of the addition of an internal quencherinto an oligonucleotide.

EXAMPLE 4

This example demonstrates the functional performance throughquantitative real time PCR (q-PCR) using probes containing the novelinternal quenchers disclosed herein.

Validated qPCR assays were used to assess the performance of thedifferent designs of fluorescence quenched probes. The primer sequencesemployed were specific for the human HPRT gene (NM_(—)00194); probe andprimer sequences and are listed below in Table 6.

TABLE 6 Sequences used in q-PCR assays SEQ ID No. Sequence Name Sequence97 HPRT Forward GACTTTGCTTTCCTTGGTCAG 98 HPRT ReverseGGCTTATATCCAACACTTCGTG 99 HPRT Probe ATGGTCAAGGTCGCAAGCTTGCTGGT

All oligonucleotides were synthesized by IDT (Integrated DNATechnologies, Coralville, Iowa). Probe oligonucleotides were HPLCpurified. Mass identity of all oligonucleotides was verified by massspectrometry. All target amplicons were cloned and sequence verified.The target plasmids were linearized by restriction endonucleasedigestion and 10 fold serial dilutions were performed to create standardcurves.

HPRT q-PCR reactions were comprised of 0.4 U Immolase DNA Polymerase(Bioline, Taunton, Mass.), 0.8 mM dNTP mix, 3 mM MgCl₂, and primer/probeconcentrations at 200 nM each. Q-PCR reactions were performed on a RocheLightcycler® 480 platform. Plasmid copy number standards were run intriplicate starting with 2×10⁷ down to 2×10² (10 fold increments). Thethermocycling profile used was 95^(10:00)−(95^(0:15)−60^(1:00))×40. Dataanalysis was performed using software supplied by the manufacturer.

Effects of placement of internal FQ (iFQ). A series of oligonucleotideprobes having a 5′-FAM reporter dye and an internal iFQ quencher weresynthesized using the HPRT probe sequence varying the relative placementof the iFQ group. In one set the iFQ group was placed as a basesubstitution (replacing a base within the sequence). In another set theiFQ group was placed as a base insertion between residues. The distanceof the iFQ from the 5′-dye was varied including 6, 8, 10, or 12positions from the 5′-end. Henceforth, a modification placed as aninsertion at position 6 is indicated as an “i6” and as a substitution atposition 8 is indicated as a “s8”, etc. The probes were used in qPCR asoutlined above using the HPRT primers (SEQ ID Nos. 97 and 98) and 2×10⁶copies of a cloned HPRT amplicon plasmid target. Amplification plots forthe substitution series probes (SEQ ID Nos. 100-103) are shown in FIG.2A and baseline normalized plots are shown in FIG. 2B. Amplificationplots for the insertion series (SEQ ID Nos. 104-107) are shown in FIG.3A and baseline normalized plots are shown in FIG. 3B. It is clear thatprecise placement of the iFQ group within the probe affected probecharacteristics, with changes seen in both the baseline fluorescence,magnitude of signal generation, and quantification cycle number (Cq, thecycle number where amplification signal is first detected).

Relative metrics for assessing probe quality from the amplificationplots are reported in Table 7, including the baseline fluorescencemeasured in the qPCR device (as a measure of quenching efficiency) andthe ΔR_(n), the difference in the fluorescence intensity between thestart and end of the qPCR run (as a measure of the magnitude of signalgenerated). Having low baseline fluorescence coupled with high relativeΔR_(n) signal generation leads to improved probe performance.

TABLE 7 Internal placement of iFQ in FAM labeled probes (HPRT assay)SEQ ID Baseline No. fluorescence ΔR_(n) Sequence (Substitution) 100FAM-ATGGTCAAGGT/iFQ/GCAAGCTTGCTGGT-SpC3 (s12) 3.2  8.0 101FAM-ATGGTCAAG/iFQ/TCGCAAGCTTGCTGGT-SpC3 (s10) 2.2  7.8 102FAM-ATGGTCA/iFQ/GGTCGCAAGCTTGCTGGT-SpC3 (s8) 1.7  7.0 103FAM-ATGGT/iFQ/AAGGTCGCAAGCTTGCTGGT-SpC3 (s6) 1.1  3.3Sequence (Insertion) 104 FAM-ATGGTCAAGGT/iFQ/CGCAAGCTTGCTGGT-SpC3 (i12)6.0 11.6 105 FAM-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT-SpC3 (i10) 4.0 13.7 106FAM-ATGGTCA/iFQ/AGGTCGCAAGCTTGCTGGT-SpC3 (i8) 2.0 12.5 107FAM-ATGGT/iFQ/CAAGGTCGCAAGCTTGCTGGT-SpC3 (i6) 1.7  6.8

All probe designs functioned in the above qPCR assay, thus the iFQ groupcan be used in the methods of this disclosure as either a basesubstitution or as an insertion between bases. Placing the iFQ group asa base substitution resulted in slightly better quenching than when usedas an insertion between bases, however as a group the substitutionprobes showed lower signal generation. Further, iFQ placed as a basesubstitution is slightly destabilizing (lowers T_(m)) whereas iFQ placedas a base insertion is stabilizing (increases T_(m)) (see Example 1above). Given the better signal generation and improved duplexstabilization properties, placement of the iFQ as an insertion isconsidered to be a more preferred embodiment and all subsequent exampleswill be done using only iFQ insertion probes.

The relative distance of the iFQ group from the 5′-FAM reporter dye hada significant impact on baseline fluorescence and on signal generation.In FIG. 3A, background fluorescence levels were i6<i8<i10<i12. Placingthe quencher group closer to the reporter dye reduced backgroundfluorescence. This effect was expected as FRET based quenching improveswith the proximity of the reporter and quencher. Unexpectedly, therelative placement of the iFQ group also affected the magnitude ofsignal generation during an amplification run and final functionalfluorescence (positive signal) was i10>i8>i12>>i6 (FIG. 3B).Interestingly, the i6 probes showed poor fluorescence signal for boththe insertion and substitution series. This reduced signal compromisedassay quality and delayed the Cq point for these probes, indicatinglower assay sensitivity. We hypothesize that placing the iFQ group tooclose to the fluorophore results in probes that are not fully cleaved bythe DNA polymerase during amplification (5′-nuclease assay format),resulting in decreased release of reporter dye from quencher. Peak probeperformance is realized at a point which is a compromise betweenquenching, which improves as quencher and fluorophore are placed moreclosely, and cleavage, which improves as quencher and fluorophore areseparated by a greater number of nucleic acid bases. The precise rangewhere this relationship is optimal is non-obvious and may be differentfor different reporter dye/quencher combinations. For this reporterdye/quencher combination, optimum performance of the assay was seen withi8 and i10 placement. For these probes, background fluorescence was lowand signal generation was high.

EXAMPLE 5

The following example demonstrates the efficacy of the internal quencherprobe design at varying target concentrations.

Example 4 examined the performance of 8 different probe designs using asingle concentration of target nucleic acid. In the present example, sixconcentrations of target were tested in HPRT qPCR assays comparingperformance of four of the probes from Example 4. HPRT specific probesusing internal FQ (iFQ) quencher with FAM reporter were employedincluding substitution design s6 and s10 (SEQ ID Nos. 103 and 101) andinsertion design i6 and i10 (SEQ ID Nos. 107 and 105). Amplificationreactions were run as outlined in Example 3 using input target plasmidcopy numbers of 2×10², 2×10³, 2×10⁴, 2×10⁵, 2×10⁶ and 2×10⁷. Eachreaction was run in triplicate.

FIG. 4A shows the results of the baseline adjusted substitution set andFIG. 4B shows the results for the baseline adjusted insertion set. Thereis a clear progression of the amplification plot curves corresponding tothe expected difference of ˜3.3 cycles for every 10-fold change of inputtarget nucleic acid, wherein the curves align from left to right thehighest concentration of template to the lowest concentration. Theinsertion probe set outperformed the substitution probe set at allconcentrations tested as evidenced by the improved magnitude of signalgenerated for all comparable data points. Further, the quencher i10placement outperformed the quencher i6 placement series. Subsequentexamples will therefore focus on use of the substitution i10 probedesign.

EXAMPLE 6

The following example demonstrates the use of an inserted internalquencher coupled with a second quencher positioned at the 3′-end of theprobe.

A new series of probes were synthesized targeting the HPRT gene allhaving a 5′-fluorescein reporter dye (6-FAM) and having quencherslocated at varying positions, including compositions having two quenchergroups in the same probe molecule. Internal quenchers were added asinsertions between bases. Single quencher probes (FQ quencher on the3′-terminus, FQ quencher at the i10 position) were compared with dualquencher version of the same sequences. Dual quencher probes were madeusing the FQ chemical group or the Black Hole Quencher™-1 (BHQ1, shownbelow), a different commercially available dark quencher (BiosearchTechnologies, Novato, Calif.). Table 8 lists the probe sequences tested.HPRT qPCR assays were performed as described in Example 4, using 2×10⁶copies of an HPRT amplicon-containing plasmid as target.

TABLE 8 Single and Dual Quenched Probes SEQ Baseline ID Probe fluor- No.Name Sequence escence ΔR_(n) 105 i10FQFAM-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT-SpC3 4.0 12.7 108 3/FQFAM-ATGGTCAAGGTCGCAAGCTTGCTGGT-FQ 10.5 11.0 109 i10FQ +FAM-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT-FQ 2.6 14.0 3′FQ 110 i10BHQ +FAM-ATGGTCAAG/iBHQ1/GTCGCAAGCTTGCTGGT-BHQ1 2.5 7.5 3′BHQ

These four probes were used in qPCR as outlined in Example 4 above usingthe HPRT primers (SEQ ID Nos. 97 and 98) and 2×10⁶ copies of a clonedHPRT amplicon plasmid target. Amplification plots are shown in FIG. 5Aand baseline normalized plots are shown in FIG. 5B. Relative metrics forassessing probe quality from the amplification plots are also reportedin Table 8, including the baseline fluorescence measured in the qPCRdevice (as a measure of quenching efficiency) and the ΔR_(n) seenbetween the start and end of the qPCR run (as a measure of the magnitudeof signal generated). Having low baseline fluorescence coupled with highrelative ΔR_(n) signal generation leads to improved probe performance.

The traditional probe design with a 5′-reporter dye and 3′-quencher (SEQID No. 108) showed the highest baseline fluorescence (i.e., worstquenching). The single quencher internal i10 placement probe (SEQ ID No.105) showed significantly lower baseline fluorescence and thedual-quencher probes (SEQ ID Nos. 109, 110) showed the lowest baselinefluorescence (i.e., best quenching). Thus the dual quencher probes inFIGS. 5A and 5B showed slightly better performance than the i10FQquencher alone, demonstrating that the presence of two quenchers notonly does not negatively affect their quenching properties but ratherimproves the overall quenching properties.

The relative fluorescence signal generated using the different probedesigns also varied with quencher type and placement. The three FQprobes generated a ΔR_(n) of 11-14 with the highest relative fluorescentsignal produced by the dual-quencher i10FQ-3′-FQ probe (SEQ ID No. 109).Interestingly, the same design using the alternative commercial darkquencher BHQ-1 (SEQ ID No. 110) performed significantly worse, showing aΔR_(n) of only 7.5. This probe also showed a delayed Cq value (the cyclenumber where amplification signal is first detected), indicating worsefunctional sensitivity. This clearly demonstrates that all dark quencherchemical compositions are not functionally interchangeable and that thenapthylene-azo quencher (FQ) performs in a superior fashion using themethods of this disclosure.

EXAMPLE 7

Examples 4, 5, and 6 were performed using a single probe sequencespecific for the human HPRT gene. The following example illustrates thatthe functional qPCR results detailed in Examples 4-6 are consistent whenrun using a different probe sequence, for a different target gene, usinga different thermal cycler platform and different reagents.

A new qPCR assay was employed specific for a strain of the H1N1Influenza virus (SW H1, also known as the “swine flu”). Primer and probesequences and are listed below in Table 9.

TABLE 9 Sequences used in an Influenza qPCR assay SEQ ID No.Sequence Name Sequence 111 SW H1 Forward GTGCTATAAACACCAGCCTYCCA 112SW H1 Reverse CGGGATATTCCTTAATCCTGTRGC 113 SW H1 ProbeCAGAATATACATCCAGTCACAATTGGAAAA

A set of Influenza virus H1N1 specific (SW H1) probes were synthesizedand qPCR assays were performed using the same methods as described inExample 4 except that the assays were run on an Applied Biosystems7900HT Sequence Detection System (Applied Biosystems, Foster City,Calif.) according to manufacturer's instructions, using 1X TaqMan GeneExpression Master Mix (Life Technologies, Carlsbad, Calif.), 250 nMprobe and 1000 nM primers per the Center for Disease Control (CDC)document (CEC REF# I-007-005 Protocol for Detection and Characterizationof Swine Influenza, 2009) recommendations. Assays run using differentprobe sequences and targets in the present example demonstrate that theperformance of the probes of this disclosure are not sequence dependent,instrument dependent or polymerase formulation dependent.

The assays were run in duplicate with 2×10⁶ copies of plasmid targetused. Table 10 lists the probe sequences tested. All internal quenchergroups were placed as insertions. Relative metrics for assessing probequality from the amplification plots are also reported in Table 10,including the baseline fluorescence measured in the qPCR device (as ameasure of quenching efficiency) and the ΔR_(n) seen between the startand end of the qPCR run (as a measure of the magnitude of signalgenerated). Note that the numbers reported are “relative fluorescenceunits” and that this example was performed on a different machine thanthe plots shown in Examples 4-6 above. The absolute numbers aredifferent between machines; however the relative performance of thevarious probe designs is directly comparable.

TABLE 10 Single and dual quenched influenza SW H1 assays SEQ ID No. NameSequence Baseline ΔRn 114 3′FQ FAM-CAGAATATACATCCAGTCACAATTGGAAAA-FQ 2.61.5 115 i10FQ FAM-CAGAATATA/iFQ/CATCCAGTCACAATTGGAAAA- 0.7 2.4 SpC3 116iF10Q + FAM-CAGAATATA/iFQ/CATCCAGTCACAATTGGAAAA-FQ 0.6 2.9 3′FQ 117i10BHQ1 + FAM-CAGAATATA/iBHQ1/CATCCAGTCACAATTGGAAAA- 0.45 0.8 3′BHQ1BHQ1

Amplification plots for the influenza qPCR assays are shown in FIG. 6Aand baseline normalized amplification plots are shown in FIG. 6B.Similar to the results described in Example 6 for the HPRT assays, theprobe containing the 3′FQ quencher alone did not perform as well as anyof the internal FQ containing probes. In particular, theinternal-quencher probes showed markedly lower baseline (background)fluorescence, with the dual-quencher probes having the lowestbackground. As before, the dual quencher probes outperformed the singlequencher probes, with the iFQ+3′FQ combination working the best. TheSWH1 probe is 30 bases long and represents a relatively long probesequence for use as a dual-quenched probe in hydrolysis assays, whichcontributes to the greater difference seen between 3′-quencher probesand internal quencher probes in this example compared with the HPRTprobes in Example 6 (the HPRT probe is 26 bases long). This illustratesanother benefit of the methods of this disclosure. Long probes (as arefrequently required in AT-rich target sequences) perform poorly using3′-quencher design; however probe length does not affect performance ofinternally quenched probes.

Similar to the results observed in Example 6 above, the dual-quencheri10FQ+3′FQ probe performed the best of the set tested, showing both lowbaseline fluorescence and high positive signal strength. Of note, againin the influenza probe sequence context the dual-quencher i10BHQ1+3′BHQ1probe showed low baseline fluorescence with very low signal strength andfunctioned poorly in the assay, showing a delayed Cq value relative tothe other probes.

EXAMPLE 8

The following example demonstrates the efficacy of the internal quencherprobes of the present disclosure when used with various fluorophores.

In previous examples, all probes contained a 5′-fluorescein reporter dye(6-FAM). Typically, quencher molecules perform well with a limitedsubset of reporter dyes that are matched such that the fluorophorefluorescence emission wavelength overlaps well with the absorbancewavelengths of the quencher. A dye with emission in the red region, suchas Cy5, typically requires use of a different quencher than one that isuseful with a dye that has a shorter wavelength emission spectra, suchas fluorescein. The probes in this example comprise differentfluorophores having a wide range of emission wavelengths (Table 10) anddemonstrate that the use of internal quencher probes in thedual-quencher format function well across a wide spectral range. Allprobes in the present example place the iFQ quencher at position i10 asan insertion. The dual-quencher probes of the present example are madeusing the i10FQ combined with either a 3′-FQ or with a 3′-IB RQ-n1 (alsoreferred to as “RQ”). In traditional single 3′-quencher probe format,the FQ quencher (which has a peak absorbance around 534 nm) is typicallyemployed with reporter dyes having emission in the 500-580 nm wavelengthrange. The RQ quencher (which has a bimodal peak absorbance around 610and 640 nm) is typically employed with reporter dyes having emission inthe 550-700 nm wavelength range.

TABLE 10 Fluorescent reporter dyes and their excitation and emissionwavelengths. Dye Excitation Max (nm) Emission Max (nm) 6-FAM 495 520 MAX524 557 Cy3 550 564 TEX-615 596 615 Cy5 648 668

A series of dual quencher probes specific for the human HPRT gene weresynthesized having an insertion of the FQ quencher at the i10 positionand either a 3′-FQ or a 3′-RQ quencher. Probe sequences are shown inTable 11 below. The probes were used in qPCR as described in Example 4above using the HPRT primers (SEQ ID Nos. 97 and 98) and 2×10⁶ copies ofa cloned HPRT amplicon plasmid target. The FAM and MAX probes were runusing the Applied Biosystems AB7900HT Sequence Detection platform. TheCy3 probes were run using the BIO-RAD iQ5 platform. The TEX615 and Cy5probes were run using the Roche LightCycler 480 platform.

Examples 6 and 7 demonstrated that the dual-quencher iFQ-3′FQcombination performed slightly better for FAM reporter dye probes thaniFQ alone, although the single quencher iFQ probes also performed well.Amplification plots for the fluorescein (emission 520 nm) reporter dyeprobes (SEQ ID Nos. 109 and 118) are shown in FIG. 7A and baselinenormalized plots are shown in FIG. 7B. In this comparison, performanceis nearly identical for the i10FQ-3′FQ vs. i10FQ-3′RQ probes, eventhough the RQ quencher is best suited for red wavelength dyes. Thei10FQ-3′FQ probe did, however, perform slightly better.

Amplification plots for the MAX (emission 557 nm) reporter dye probes(SEQ ID Nos. 119 and 120) are shown in FIG. 8A and baseline normalizedplots are shown in FIG. 8B. Baseline quenching was nearly identical forthe i10FQ-3′FQ vs. i10FQ-3′RQ probes, however peak signal intensity wassuperior with the i10FQ-3′FQ probe. Both designs worked well but thei10FQ-3′FQ design is preferred.

Amplification plots for the Cy3 (emission 564 nm) reporter dye probes(SEQ ID Nos. 121 and 122) are shown in FIG. 9A and baseline normalizedplots are shown in FIG. 9B. Baseline quenching was slightly lower forthe i10FQ-3′FQ vs. i10FQ-3′RQ probes, and peak signal intensity was alsoslightly superior with the i10FQ-3′FQ probe. Both designs worked wellbut the i10FQ-3′FQ design is preferred.

Amplification plots for the TEX615 (emission 615 nm) reporter dye probes(SEQ ID Nos. 123 and 124) are shown in FIG. 10A and baseline normalizedplots are shown in FIG. 10B. Baseline quenching was slightly lower forthe i10FQ-3′RQ vs. i10FQ-3′FQ probes, however peak signal intensity wassuperior with the i10FQ-3′FQ probe. Both designs worked well but thei10FQ-3′FQ design is preferred.

Amplification plots for the Cy5 (emission 668 nm) reporter dye probes(SEQ ID Nos. 125 and 126) are shown in FIG. 11A and baseline normalizedplots are shown in FIG. 11B. Baseline quenching was lower for thei10FQ-3′RQ vs. i10FQ-3′FQ probes; however peak signal intensity wasidentical for both designs. Both designs worked well but the i10FQ-3′RQdesign showed lower baseline fluorescence.

The relative metrics for assessing probe quality from the amplificationplots are also reported in Table 12, including the baseline fluorescencemeasured in the qPCR device (as a measure of quenching efficiency) andthe ΔR_(n) seen between the start and end of the qPCR run (as a measureof the magnitude of signal generated). Having low baseline fluorescencecoupled with high relative ΔR_(n) signal generation leads to improvedprobe performance. Note that different real-time PCR machines wereemployed for different probe pairs (see above) and that the arbitraryfluorescence units reporting fluorescence intensity vary betweenplatforms.

TABLE 12 Dual-quencher probes with various reporter dyes and an internali10FQ with either 3′-FQ or 3′-RQ SEQ ID Baseline No. Name Sequencefluorescence ΔR_(n) 109 FAM FAM-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 2.9 14i10FQ-3′FQ FQ 118 FAM FAM-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 3.1 13.8i10FQ-3′RQ RQ 119 MAX MAX-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 0.2 2.1i10FQ-3′FQ FQ 120 MAX MAX-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 0.2 1.9i10FQ-3′RQ RQ 121 Cy3 Cy3-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 350 1500i10FQ-3′FQ FQ 122 Cy3 Cy3-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 250 1300i10FQ-3′RQ RQ 123 TEX TEX-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 0.8 3.4i10FQ-3′FQ FQ 124 TEX TEX-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 0.7 2.4i10FQ-3′RQ RQ 125 Cy5 Cy5-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 0.4 1.5i10FQ-3′FQ FQ 126 Cy5 Cy5-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT- 0.2 1.5i10FQ-3′RQ RQ

The use of an internal quencher paired with a 3′-quencher in the abovedual-quencher probes enables use of a single quencher compound (FQ) inprobes with reporter dyes ranging from FAM to Cy5. This novel formatthus permits use of a quencher with dyes that it is not suitable to workwith when used in the traditional 3′-quencher alone probe design format.

EXAMPLE 9

The following example demonstrates that placement of the internal iFQinsertion can vary with the Cy5 reporter dye.

The probes studied in Example 8 all employed the FQ quencher placed asan insertion at position i10. This example compares function of adual-quenched Cy5 probes having i10FQ vs. i12FQ.

Probe sequences are shown in Table 13 below. The probes were used inqPCR as described in Example 4 above using the HPRT primers (SEQ ID Nos.97 and 98) and 2×10⁷ to 2×10² copies of a cloned HPRT amplicon plasmidtarget. The Cy5 probes were run using the Roche LightCycler 480platform.

TABLE 13 Dual-quencher probes with Cy5 reporter dye comparing i10FQ vs. i12FQ SEQ ID No. Name Sequence 125 Cy5Cy5-ATGGTCAAG/iFQ/GTCGCAAGCTTGCTGGT-FQ i10FQ-3′FQ 127 Cy5Cy5-ATGGTCAAGGT/iFQ/CGCAAGCTTGCTGGT-RQ i12FQ-3′FQThe baseline adjusted amplification plot showing superimposed traces ofall 6 dilution curves for both probes is shown in FIG. 12. Peak signalintensity was slightly superior for the i10FQ-3′FQ probe than for thei12FQ-3′FQ probe, however both probes performed equally well inquantitative detection of the target standard curve dilution set.Precise Cq values measured for each probe/target dilution are shown inTable 14 below, where it can be seen that the Cq values are nearlyidentical between the two probes.

TABLE 14 Cq values comparing sensitivity of i10FQ-3′FQ vs. i12FQ-3-FQfor an HPRT assay using Cy5 reporter dye Copy Number CY5 i10FQ-3′FQ CY5i12FQ-3′FQ 2E7 14.2 14.4 2E6 17.5 17.4 2E5 20.9 21.0 2E4 24.8 24.6 2E328.0 27.9 2E2 31.6 32.4

Although there may be range in location for placement of the iFQinsertion for different probes, the preferred location may vary withdifferent reporter dyes. More importantly, there exists some flexibilityin placement such that iFQ group place anywhere in the i8-i12 rangeshould work well.

EXAMPLE 10

This example demonstrates the synthesis of a phosphoramidite useful foroligonucleotide synthesis that is derivatized with a quencher of thepresent disclosure. The synthetic method is shown below in Schemes 1 and2.

Mono-DMT-phenyl diethanolamine (2): A solution of 10 g of phenyldiethanolamine in 100 mL of pyridine was mixed for 3-4 h at roomtemperature with a solution of 6 g dimethoxytrityl-chloride (DMT-Cl) in150 mL of a 98:2 dichloromethane/pyridine solution. The reaction mixturewas concentrated to dryness under vacuum. The residue was dissolved in200 mL of ethyl acetate, washed with two portions of 100 mL of deionizedwater, and the organic layer dried over Na₂SO₄. The organic solution wasconcentrated and purified by column chromatography using a 300 g ofsilica gel column developed with 30/65/5 ethylacetate/hexanes/triethylamine to yield 5.25 g (20% yield) ofmono-DMT-phenyl diethanolamine. TLC: R_(f) 0.55(EtAc/hexanes/Et₃N—40/55/5). ¹H NMR (CDCl₃) δ 7.38 (d, J=8 Hz, 2H), 7.27(d, J=8 Hz, 4H), 7.38 (d, J=8 Hz, 2H), 7.24-7.12 (m, 6H), 6.76 (d, J=8Hz, 4H), 6.66 (d, J=8 Hz, 2H), 3.74 (s, 6H), 3.74 (t, J=7.5 Hz, 2H),3.54 (t, J=7.5 Hz, 2H), 3.51 (t, J=7.5 Hz, 2H), 3.33 (t, J=7.5 Hz, 2H),2.23 (br. s, 1H).

Mono-DMT-4-(1-nitro-4-naphthylazo)-N,N-diethanolaniline (3): Coldconcentrated HCl (17 mL) was added dropwise at 0° C. over 15 min to asuspension of 4-nitro-1-naphthylamine (2 g) in cold water (6 mL) at 0°C. Then NaNO₂ (1.6 g) in cold water (4 mL) was added dropwise at 0° C.over 15 min and the 4-nitro-1-naphthylamine dissolved upon stirring.LiBF₄ (1.38 g) in H₂O (3 mL) was added dropwise at 0° C. The reactionmixture was stirred at 0° C. for 30 min. A brownish yellow powder (3.08g) of naphthyl-1-nitro-4-tetrafluoroborate azonium salt (1) was obtainedafter filtering and rinsing the solution with cold water, methanol, andether. A solution of 4 g of mono-DMT-phenyl diethanolamine (2) in 50 mLof dimethylsulfoxide (DMSO) was added with stirring at 10-15° C. over10-15 min to a chilled solution of 2.8 g of azonium salt (1) in 50 mL ofDMSO at 10-15° C. in a water bath. After an additional 15 min ofstirring, 3 mL of triethylamine was added to the reaction mixturefollowed by 100 mL of ethyl acetate. The reaction mixture was washedwith 3×30 mL of deionized water and the organic layer was dried overNa₂SO₄. The solvent was removed and product was purified by columnchromatography with 300 g of silica gel to provide 1.8 g ofmono-DMT-4-(1-nitro-4-naphtylazo)-N,N-diethanolaniline (3). TLC: R_(f)0.65 (DCM/Et₃N—80/20). ¹H NMR (CDCl₃) δ 9.04 (d, J=8.4 Hz, 1H), 8.68 (d,J=8.4 Hz, 1H), 8.34 (d, J=8.4 Hz, 1H), 7.96 (d, J=9.2 Hz, 2H), 7.81-7.71(m, 3H), 7.39 (d, J=8 Hz, 2H), 7.27 (d, J=8 Hz, 4H), 7.24-7.19 (m, 3H),6.78 (d, J=8 Hz, 4H), 6.77 (d, J=8 Hz, 2H), 3.88 (t, J=7.5 Hz, 2H), 3.75(s, 6H), 3.78-368 (m, 4H), 3.47 (t, J=7.5 Hz, 2H), 1.57 (br. s, 1H).

Mono-DMT-4-(1-nitro-4-naphthylazo)-N,N-diethanolaniline phosphoramidite(4): A solution of 0.2 ml ofN,N-diisopropylamino-cyanoethyl-phosphoramidolchloride was stirred intoa solution of 0.3 g of alcohol (3) in 20 mL of anhydrous THF and 1 mL oftriethylamine for 5 min at 0-5° C. After 15 min of additional stirringthe reaction mixture was warmed to room temperature. The solvent wasevaporated under a vacuum and the residue purified by columnchromatography through 50 g of silica gel (EtOAc/PE/TEA:10/85/5-40/55/5). TLC: R_(f) 0.65 (DCM/Et₃N—80/20). ¹H NMR (CDCl₃) δ9.05 (d, J=8.4 Hz, 1H), 8.68 (d, J=8.4 Hz, 1H), 8.34 (d, J=8.4 Hz, 1H),7.96 (d, J=9.2 Hz, 2H), 7.81-7.71 (m, 3H), 7.39 (d, J=8 Hz, 2H), 7.27(d, J=8 Hz, 4H), 7.24-7.19 (m, 3H), 6.78 (d, J=8 Hz, 4H), 6.76 (d, J=8Hz, 2H), 3.85-3.75 (m, J=7.5 Hz, 4H), 3.76 (s, 6H), 3.70 (t, J=7.5 Hz,2H), 3.41 (t, J=7.5 Hz, 2H), 2.58 (t, J=8.0 Hz, 2H), 1.20 (s, 3H), 1.18(s, 3H), 1.17 (s, 3H), 1.15 (s, 3H). ³¹P NMR δ 148.39.

The phosphoramidite (4) can be added to an oligonucleotide duringsynthesis using standard phosphoramidite oligonucleotide synthetictechniques.

EXAMPLE 11

The following example demonstrates the utility of the disclosedinsertions in a molecular beacon probe.

All Molecular Beacon oligonucleotides were synthesized and purified byIntegrated DNA Technologies, Inc. Molecular beacon oligonucleotides at200 nM were incubated in a buffer consisting of 16.0 mM (NH₄)₂SO₄, 67.0mM Tris-HCl pH 8.3, 0.01% Tween-20 in the presence and absence of a 1000nM complementary oligonucleotide. The samples were incubated at 30° C.in a CFX884 Real Time System (BioRad, Hercules, Calif.), and subjectedto an increase in temperature from 30-95° C. at a ramp rate of 1°C./min, with fluorescence measurements taken at each degree interval.First derivative analysis of the fluorescence curves yielded the meltingtemperature of the beacon alone (stem T_(m)) and the melting temperatureof the probe plus complement (loop T_(m)). Each sample independently wasmeasured a minimum of three times.

Table 15 lists the sequences that were synthesized and tested. The “RQ”is IBRQn-1.

TABLE 15 Molecular beacon sequences SEQ ID No. Name Sequence 128 beaconCGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG 129 complementTTACATGAAGCCCACTCCTTGTCTATC 130 3′ Dabcyl/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG/3Dabcyl/ 131 3′ BHQ-1/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG/3BHQ-1/ 132 3′ FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG/3FQ/ 133 FQ0-FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG/FQ/FQ/ 134 FQ1-FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGC/FQ/G/3FQ/ 135 FQ2-FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCG/FQ/CG/3FQ/ 136 FQ3-FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATC/FQ/GCG/3FQ/ 137 FQ4-FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGAT/FQ/CGCG/3FQ/ 138 FQ5-FQ/56-FAM/CGCGATCAGACAAGGAGIGGGCTTCATGGA/FQ/TCGCG/3FQ/ 139 FQ6-FQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGG/FQ/ATCGCG/3FQ/ 140 3′ RQSp/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG/3RQSp/ 141 FQ0-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGCG/FQ//3RQSp/ 142 FQ1-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCGC/FQ/G/3RQSp/ 143 FQ2-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATCG/FQ/CG/3RQSp/ 144 FQ3-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGATC/FQ/GCG/3RQSp/ 145 FQ4-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGAT/FQ/CGCG/3RQSp/ 146 FQ5-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGGAJFQ/TCGCG/3RQSp/ 147 FQ6-RQ/56-FAM/CGCGATCAGACAAGGAGTGGGCTTCATGG/FQ/ATCGCG/3RQSp/

Table 16 lists the results of the assay for each beacon.

TABLE 16 Quencher Stem T_(m) Stem ΔT_(m) Loop T_(m) Loop ΔT_(m) 3′Dabcyl66 63 3′BHQ-1 70 62.7 3′FQ 70 — 63 — FQ0-FQ 72 2 62 −1 FQ1-FQ 68 −2 63 0FQ2-FQ 70 0 62 −1 FQ3-FQ 71 1 62 −1 FQ4-FQ 72 2 62 −1 FQ5-FQ 74 4 63 0FQ6-FQ 75.3 5.3 64 1 3′RQSp 74 — 61 — FQ0-RQ 74 0 63 2 FQ1-RQ 70 −4 62 1FQ2-RQ 71 −3 61 0 FQ3-RQ 71 −3 61 0 FQ4-RQ 73 −1 62 1 FQ5-RQ 76 2 62 1FQ6-RQ 77 3 61.3 0.3

The stability is improved when the FQ quencher is placed internallyclose to the 3′ quencher, particularly with a 3′ RQ quencher.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of this disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.The terms “comprising,” “having,” “including,” and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the variousinventions disclosed herein and does not pose a limitation on the scopeof any inventions unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice of the invention.

Preferred embodiments of various inventions are described herein,including the best mode known to the inventors for carrying out thevarious inventions. Variations of those preferred embodiments may becomeapparent to those of ordinary skill in the art upon reading theforegoing description. The inventors expect skilled artisans to employsuch variations as appropriate, and the inventors intend for the variousinventions to be practiced otherwise than as specifically describedherein. Accordingly, this disclosure includes all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the disclosure unless otherwise indicated herein orotherwise clearly contradicted by context.

What is claimed is:
 1. A method of detecting a target oligonucleotide ina sample, comprising: contacting the sample with a compositioncomprising a first oligonucleotide having the structure5′-Y₁-L₁-X-L₂-Y₂-3′, wherein Y₁ comprises a sequence of DNA or RNAnucleotides, including a first nucleotide N₁ having a 3′ phosphatecovalently linked to L₁; Y₂ comprises a sequence of DNA or RNAnucleotides, including a second nucleotide N₂ having a 5′ phosphatecovalently linked to L₂; L₁ and L₂ each independently are a direct bondor a C₁-C₇ alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl,aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxylgroup; X is

R₁ is a hydrogen or a C₁-C₈ alkyl; M is

L₃ is a direct bond or a C₁-C₈ alkyl, alkenyl, alkenyl, heteroalkyl,substituted alkyl, cycloalkyl, or alkoxyl; R₂-R₆ each independently area hydrogen, an alkenyl, a heteroalkyl, a substituted alkyl, an aryl, aheteroaryl, a substituted aryl, a cycloalkyl, an alkylaryl, an alkoxyl,an electron withdrawing group, or an electron donating group, and one ofR₂-R₆ comprises

wherein P is

R₇-R₉ each independently are a hydrogen, an alkoxyl, an alkyl, analkylamino, an arylamino, a cycloalkyl, a heteroalkoxyl, a heteroalkyl,or an amino; and R₁₀-R₁₃ each independently are a hydrogen, a nitro, acyano, a carboxylate, a sulfonyl, a sulfamoyl, an alkenyl, an alkynyl,an amino, an aryl, a heteroaryl, a biaryl, a bialkenyl, a bialkenyl, analkoxycarbonyl or a carbamoyl; wherein the first oligonucleotide isadapted to hybridize to a second oligonucleotide having the structure3′-Y₃-Y₄-5′; wherein Y₃ comprises a sequence of DNA or RNA nucleotidesincluding a third nucleotide N₃; and wherein Y₄ comprises a sequence ofDNA or RNA nucleotides including a fourth nucleotide N₄ that is directlyattached to nucleotide N₃; wherein if the first oligonucleotidehybridizes to the second oligonucleotide, N₁ base pairs with N₃ and N₂base pairs with N₄ to form a duplex having a T_(m) that is greater thanthe T_(m)of a duplex formed between the second oligonucleotide and athird oligonucleotide having the structure 5′-Y₁-Y₂-3′; wherein thefirst oligonucleotide is also adapted to hybridize to the targetoligonucleotide; wherein the first oligonucleotide is labeled with afluorophore; and wherein fluorescence of the fluorophore is reduced byfluorescence resonance energy transfer to the quencher or by groundstate quenching by the quencher when the first oligonucleotide is nothybridized to the target oligonucleotide; and detecting an increase influorescence indicating the presence of the target oligonucleotide inthe sample.
 2. The method of claim 1, wherein fluorescence is reduced byfluorescence resonance energy transfer when the first oligonucleotide isnot hybridized to the target oligonucleotide.
 3. The method of claim 1,wherein fluorescence is reduced by ground state quenching when the firstoligonucleotide is not hybridized to the target oligonucleotide.
 4. Themethod of claim 1, wherein fluorescence is reduced by both fluorescenceresonance energy transfer and ground state quenching when the firstoligonucleotide is not hybridized to the target oligonucleotide.
 5. Themethod of claim 1, wherein the increase in fluorescence arises fromcleavage of the target oligonucleotide.
 6. The method of claim 1,wherein the oligonucleotide forms a random-coil conformation when theoligonucleotide is unhybridized, such that the fluorescence of thefluorophore is reduced.
 7. The method of claim 1, wherein theoligonucleotide comprises a self-complimentary sequence and wherein thequencher and the fluorophore are attached to the oligonucleotide suchthat the fluorescence of the fluorophore is quenched by the quencherwhen the nucleic acid polymer undergoes intramolecular base pairing. 8.The method of claim 1, wherein the method is used in a PCR reactionwherein synthesis of PCR product results in an increase in fluorescence.9. The method of claim 1, wherein the target nucleotide is the secondnucleotide.
 10. The method of claim 1, wherein R₉ is


11. The method of claim 1, wherein the fluorophore is6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX); 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, 2-p-toluidinyl-6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarindye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5 (Cy5.5),3-1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed), a BODIPY™ dye, benzoxaazole, stilbene or pyrene.
 12. The method ofclaim 1, wherein the oligonucleotide is labeled with a second quencher.13. The method of claim 12, wherein the second quencher is dabcyl,Eclipse® quencher, BHQ1, BHQ2 and BHQ3, Iowa Black® FQ, Iowa Black®RQ-n1 or Iowa Black® RQ-n2.
 14. The method of claim 13, wherein thesecond quencher is Iowa Black® FQ, Iowa Black® RQ-n1 or Iowa Black®RQ-n2.
 15. A method of detecting a target oligonucleotide in a sample,comprising: contacting the sample with a composition comprising a firstoligonucleotide having the structure 5′-Y₁-L₁-X-L₂-Y₂-3′, wherein Y₁comprises a sequence of DNA or RNA nucleotides, including a firstnucleotide N₁ having a 3′ phosphate covalently linked to L₁; Y₂comprises a sequence of DNA or RNA nucleotides, including a secondnucleotide N₂ having a 5′ phosphate covalently linked to L₂; L₁ and L₂each independently are a direct bond or a C₁-C₇ alkyl, alkynyl, alkenyl,heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl,cycloalkyl, alkylaryl, or alkoxyl group; X is

R₁ is a hydrogen or a C₁-C₈ alkyl; and M is

L₃ is a direct bond or a C₁-C₈ alkyl, alkenyl, alkenyl, heteroalkyl,substituted alkyl, cycloalkyl, or alkoxyl; R₂-R₆ each independently area hydrogen, an alkyl, an alkenyl, a heteroalkyl, a substituted alkyl, anaryl, a heteroaryl, a substituted aryl, a cycloalkyl, an alkylaryl, analkoxyl, an electron withdrawing group, or an electron donating group,and one of R₂-R₆ comprises

wherein P is

and R₁₄-R₁₉ each independently are a hydrogen, an alkyl, a heteroalkyl,an aryl, a heteroaryl, an electron withdrawing group, or a five or sixmembered ring structure formed from the R1, R2 pair, the R₃, R₄ pair,the R₄, R₅ pair, or the R₅, R₆ pair; wherein the first oligonucleotideis adapted to hybridize to a second oligonucleotide having the structure3′-Y₃-Y₄-5′; wherein Y₃ comprises a sequence of DNA or RNA nucleotidesincluding a third nucleotide N₃; and wherein Y₄ comprises a sequence ofDNA or RNA nucleotides including a fourth nucleotide N₄ that is directlyattached to nucleotide N₃; wherein if the first oligonucleotidehybridizes to the second oligonucleotide, N₁ base pairs with N₃ and N₂base pairs with N₄ to form a duplex having a T_(m) that is greater thanthe T_(m) of a duplex formed between the second oligonucleotide and athird oligonucleotide having the structure 5′-Y₁-Y₂-3′; wherein thefirst oligonucleotide is also adapted to hybridize to the targetoligonucleotide; wherein the first oligonucleotide is labeled with afluorophore; and wherein fluorescence of the fluorophore is reduced byfluorescence resonance energy transfer to the quencher or by groundstate quenching by the quencher when the first oligonucleotide is nothybridized to the target oligonucleotide; and detecting an increase influorescence indicating the presence of the target oligonucleotide inthe sample.
 16. The method of claim 15, wherein fluorescence is reducedby fluorescence resonance energy transfer when the first oligonucleotideis not hybridized to the target oligonucleotide.
 17. The method of claim15, wherein fluorescence is reduced by ground state quenching when thefirst oligonucleotide is not hybridized to the target oligonucleotide.18. The method of claim 15, wherein fluorescence is reduced by bothfluorescence resonance energy transfer and ground state quenching whenthe first oligonucleotide is not hybridized to the targetoligonucleotide.
 19. The method of claim 15, wherein the increase influorescence arises from cleavage of the target oligonucleotide.
 20. Themethod of claim 15, wherein the oligonucleotide forms a random-coilconformation when the oligonucleotide is unhybridized, such that thefluorescence of the fluorophore is reduced.
 21. The method of claim 15,wherein the oligonucleotide comprises a self-complimentary sequence andwherein the quencher and the fluorophore are attached to theoligonucleotide such that the fluorescence of the fluorophore isquenched by the quencher when the nucleic acid polymer undergoesintramolecular base pairing.
 22. The method of claim 15, wherein themethod is used in a PCR reaction wherein synthesis of PCR productresults in an increase in fluorescence.
 23. The method of claim 15,wherein the target nucleotide is the second nucleotide.
 24. The methodof claim 15, wherein the fluorophore is 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX); 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, 2-p-toluidinyl -6-naphthalene sulfonate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), a coumarindye, an acridine dye, indodicarbocyanine 3 (Cy3), indodicarbocyanine 5(Cy5), indodicarbocyanine 5.5 (Cy5.5),3-1-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed), a BODIPY™ dye, benzoxaazole, stilbene or pyrene.
 25. The method ofclaim 15, wherein the oligonucleotide is labeled with a second quencher.26. The method of claim 25, wherein the second quencher is dabcyl,Eclipse® quencher, BHQ1, BHQ2 and BHQ3, Iowa Black® FQ, Iowa Black®RQ-n1 or Iowa Black® RQ-n2.
 27. The method of claim 26, wherein thesecond quencher is Iowa Black® FQ, Iowa Black® RQ-n1 or Iowa Black®RQ-n2.